Methods for the preparation of biologically active compounds in nanoparticulate form

ABSTRACT

A method for producing a composition comprising nanoparticles of a biologically active compound.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/016,443, filed Jun. 22, 2018, which is a continuation of U.S.application Ser. No. 14/461,594, filed Aug. 18, 2014, which is acontinuation of U.S. application Ser. No. 12/306,948, filed Dec. 7,2009, (U.S. Pat. No. 8,808,751), which is a U.S. national stage under 35USC § 371 of International Application Number PCT/AU07/00910, filed Jun.29, 2007, which claims priority to AU Application No. 2006903527, filedJun. 30, 2006 and U.S. Provisional Application No. 60/915,955, filed May4, 2007, the entire contents of which applications are herebyincorporated by reference.

FIELD OF THE INVENTION

The present invention relates to methods for the preparation ofbiologically active compounds in nanoparticulate form. The inventionalso relates to biologically active compounds in nanoparticulate formproduced by said methods, to compositions comprising such compounds, tomedicaments produced using said biologically active compounds innanoparticulate form and/or compositions, and to methods of treatment ofan animal, including man, using a therapeutically effective amount ofsaid biologically active compounds administered by way of saidmedicaments.

BACKGROUND

Poor bioavailability is a significant problem encountered in thedevelopment of therapeutic compositions, particularly those compoundscontaining a biologically active compound that is poorly soluble inwater at physiological pH. An active compound's bioavailability is thedegree to which the active compound becomes available to the targettissue in the body after systemic administration through, for example,oral or intravenous means. Many factors affect bioavailability,including the form of dosage and the solubility and dissolution rate ofthe active compound.

Poorly and slowly water-soluble compounds tend to be eliminated from thegastrointestinal tract before being absorbed into the circulation. Inaddition, poorly soluble active agents tend to be disfavored or evenunsafe for intravenous administration due to the risk of particles ofagent blocking blood flow through capillaries.

It is known that the rate of dissolution of a particulate drug willincrease with increasing surface area. One way of increasing surfacearea is decreasing particle size. Consequently, methods of making finelydivided or sized drugs have been studied with a view to controlling thesize and size range of drug particles for pharmaceutical compositions.

For example, dry milling techniques have been used to reduce particlesize and hence influence drug absorption. However, in conventional drymilling the limit of fineness is reached generally in the region ofabout 100 microns (100,000 nm), at which point material cakes on themilling chamber and prevents any further diminution of particle size.Alternatively, wet grinding may be employed to reduce particle size, butflocculation restricts the lower particle size limit to approximately 10microns (10,000 nm). The wet milling process, however, is prone tocontamination, thereby leading to a bias in the pharmaceutical artagainst wet milling. Another alternative milling technique, commercialairjet milling, has provided particles ranging in average size from aslow as about 1 to about 50 microns (1,000-50,000 nm).

There are several approaches currently used to formulate poorly solubleactive agents. One approach is to prepare the active agent as a solublesalt. Where this approach cannot be employed, alternate (usuallyphysical) approaches are employed to improve the solubility of theactive agent. Alternate approaches generally subject the active agent tophysical conditions that change the agent's physical and or chemicalproperties to improve its solubility. These include process technologiessuch as micro-ionisation, modification of crystal or polymorphicstructure, development of oil based solutions, use of co-solvents,surface stabilizers or complexing agents, micro-emulsions, supercritical fluid and production of solid dispersions or solutions. Morethan one of these processes may be used in combination to improveformulation of a particular therapeutic compound.

These techniques for preparing such pharmaceutical compositions tend tobe complex. By way of example, a principal technical difficultyencountered with emulsion polymerization is the removal of contaminants,such as unreacted monomers or initiators (which may have undesirablelevels of toxicity), at the end of the manufacturing process.

Another method of providing reduced particle size is the formation ofpharmaceutical drug microcapsules, which techniques include micronizing,polymerisation and co-dispersion. However, these techniques suffer froma number of disadvantages including at least the inability to producesufficiently small particles such as those obtained by milling, and thepresence of co-solvents and/or contaminants such as toxic monomers whichare difficult to remove, leading to expensive manufacturing processes.

Over the last decade, intense scientific investigation has been carriedout to improving the solubility of active agents by converting theagents to ultra fine powders by methods such as milling and grinding.These techniques may be used to increase the dissolution rate of aparticulate solid by increasing the overall surface area and decreasingthe average particle size.

U.S. Pat. No. 6,634,576 discloses examples of wet-milling a solidsubstrate, such as a pharmaceutically active compound, to produce a“synergetic co-mixture”.

International Patent Application PCT/AU2005/001977 (NanoparticleComposition(s) and Method for Synthesis Thereof) describes, inter alia,a method comprising the step of contacting a precursor compound with aco-reactant under mechanochemical synthesis conditions wherein asolid-state chemical reaction between the precursor compound and theco-reactant produces therapeutically active nanoparticles dispersed in acarrier matrix. Mechanochemical synthesis, as discussed in InternationalPatent Application PCT/AU2005/001977, refers to the use of mechanicalenergy to activate, initiate or promote a chemical reaction, a crystalstructure transformation or a phase change in a material or a mixture ofmaterials, for example by agitating a reaction mixture in the presenceof a milling media to transfer mechanical energy to the reactionmixture, and includes without limitation “mechanochemical activation”,“mechanochemical processing”, “reactive milling”, and related processes.

The present invention provides methods for the preparation ofbiologically active compounds in nanoparticulate form, which amelioratesome of the problems attendant with prior technologies, or provides analternative thereto.

As an example of the need for such novel compounds and methods forsynthesizing them, consider osteoporosis. Osteoporosis describes a groupof diseases which arises from diverse etiologies, but which arecharacterized by the net loss of bone mass per unit volume. Theconsequence of this loss of bone mass and resulting bone fracture is thefailure of the skeleton to provide adequate support for the body. One ofthe most common types of osteoporosis is associated with menopause. Mostwomen lose from about 20% to about 60% of the bone mass in thetrabecular compartment of the bone within 3 to 6 years after thecessation of menses. This rapid loss is generally associated with anincrease of bone resorption and formation. However, the resorptive cycleis more dominant and the result is a net loss of bone mass. Osteoporosisis a common and serious disease among postmenopausal women.

The most generally accepted method for the treatment of postmenopausalosteoporosis is estrogen replacement therapy. Although therapy isgenerally successful, patient compliance with the therapy is low,primarily because estrogen treatment frequently produces undesirableside effects. An additional method of treatment would be theadministration of a bisphosphonate compound, such as, for example,Fosamax™ (Merck & Co., Inc.).

Throughout premenopausal time, most women have less incidence ofcardiovascular disease than men of the same age. Following menopause,however, the rate of cardiovascular disease in women slowly increases tomatch the rate seen in men. This loss of protection has been linked tothe loss of estrogen and, in particular, to the loss of estrogen'sability to regulate the levels of serum lipids. The nature of estrogen'sability to regulate serum lipids is not well understood, but evidence todate indicates that estrogen can up regulate the low density lipid (LDL)receptors in the liver to remove excess cholesterol. Additionally,estrogen appears to have some effect on the biosynthesis of cholesterol,and other beneficial effects on cardiovascular health.

It has been reported in the literature that serum lipid levels inpostmenopausal women having estrogen replacement therapy return toconcentrations found in the premenopausal state. Thus, estrogen wouldappear to be a reasonable treatment for this condition. However, theside effects of estrogen replacement therapy are not acceptable to manywomen, thus limiting the use of this therapy. An ideal therapy for thiscondition would be an agent which regulates serum lipid levels in amanner analogous to estrogen, but which is devoid of the side effectsand risks associated with estrogen therapy.

A number of structurally unrelated compounds are capable of interactingwith the estrogen receptor and producing unique in vivo profiles.Compounds with in vivo profiles typical of a “pure” antagonist (forexample, ICI 164,384) or of a relatively “pure” agonist (for example,17β-estradiol) represent opposite ends of a spectrum in thisclassification. Between these two extremes lie the SERMs (“selectiveestrogen receptor modulator”), characterized by clinical and/orpreclinical selectivity as full or partial agonists in certain desiredtissues (for example, bone), and antagonists or minimal agonists inreproductive tissues. Within this pharmacologic class, individual SERMsmay be further differentiated based on profiles of activity inreproductive tissues.

Raloxifene, a second generation SERM, displays potentially usefulselectivity in uterine tissue with apparent advantages overtriphenylethylene-based estrogen receptor ligands. As such, raloxifeneappears to be well-suited at least for the treatment of postmenopausalcomplications, including osteoporosis and cardiovascular disease. It isanticipated that, as further advances are made in the pharmacology andmolecular biology of estrogen receptor active agents, furthersubclassifications of SERMs may evolve in the future along with anincreased understanding of the therapeutic utility of these novelclasses of estrogenic compounds.

The advancement of raloxifene has been hampered by its physicalcharacteristics, particularly low solubility, which affectsbioavailability. Accordingly, any improvement in the physicalcharacteristics of raloxifene would potentially offer more beneficialtherapies. In particular, it would be a significant contribution to theart to provide forms of raloxifene which have increased solubility,methods of preparation of such forms, pharmaceutical formulationscomprising such forms, and methods of use of such formulations.

Although the background to the present invention is discussed in thecontext of improving the bioavailability of compounds that are poorly orslowly water soluble, the applications of the methods of the presentinvention are not limited to such, as is evident from the followingdescription of the invention.

Further, although the background to the present invention is largelydiscussed in the context of improving the bioavailability of therapeuticor pharmaceutical compounds, the applications of the methods of thepresent invention are clearly not limited to such. For example, as isevident from the following description, applications of the methods ofthe present invention include but are not limited to: veterinarytherapeutic applications and agricultural chemical applications, such aspesticide and herbicide applications.

SUMMARY OF THE INVENTION

The present invention is directed to the unexpected discovery thatbiologically active compounds in nanoparticulate form can be produced bydry milling solid biologically active compound together with a millablegrinding compound, such that the resulting nanoparticulate biologicallyactive compound dispersed in milled grinding compound resistsreagglomeration.

Thus, in one aspect, the present invention comprises a method forproducing a biologically active compound in nanoparticulate form, themethod comprising the step of:

-   -   dry milling a mixture of a solid biologically active compound        and a millable grinding compound, in a mill comprising a        plurality of milling bodies, to produce a solid dispersion or        solution comprising nanoparticulate biologically active compound        dispersed in at least partially milled grinding compound.

The term millable means that the grinding compound is capable of beingphysically degraded under the dry milling conditions of the method ofthe invention. In one embodiment of the invention, the milled grindingcompound is of a comparable particle size to the nanoparticulatebiologically active compound.

Without wishing to be bound by theory, it is believed that the physicaldegradation of the millable grinding compound affords the advantage ofthe invention by acting as a more effective diluent than grindingcompounds of a larger particle size.

In a highly preferred form, the grinding compound is harder than thebiologically active compound, and is thus capable of physicallydegrading such under the dry milling conditions of the invention. Againwithout wishing to be bound by theory, under these circumstances it isbelieved that the millable grinding compound affords the advantage ofthe present invention through a second route, with the smaller particlesof grinding compound produced under the dry milling conditions enablingthe production of smaller particles of biologically active compound.

The solid dispersion or solution may then be separated from the millingbodies and removed from the mill.

In a preferred aspect, the grinding compound is separated from thedispersion or solution. In one aspect, where the grinding compound isnot fully milled, the unmilled grinding compound is separated from thenanoparticulate biologically active compound. In a further aspect, atleast a portion of the milled grinding compound is separated from thenanoparticulate biologically active compound.

The milling bodies are essentially resistant to fracture and erosion inthe dry milling process.

The quantity of the grinding compound relative to the quantity ofbiologically active compound in nanoparticulate form, and the extent ofphysical degradation of the grinding compound, is sufficient to inhibitreagglomeration of the biologically active compound in nanoparticulateform. The grinding compound is not chemically reactive with thepharmaceutical compound under the milling conditions of the invention.

In additional aspects, the present invention also relates tobiologically active compounds in nanoparticulate form produced by saidmethods, to compositions comprising said compounds, to medicamentsproduced using said biologically active compounds in nanoparticulateform and/or said compositions, and to methods of treatment of an animal,including man, using a therapeutically effective amount of saidbiologically active compounds administered by way of said medicaments.

Medicaments of the invention may comprise only the biologically activecompounds in nanoparticulate form or, more preferably, the biologicallyactive compounds in nanoparticulate form may be combined with one ormore pharmaceutically acceptable carriers, as well as any desiredexcipients or other like agents commonly used in the preparation ofmedicaments.

While the method of the present invention has particular application inthe preparation of poorly water-soluble biologically active compounds innanoparticulate form, the scope of the invention is not limited thereto.For example, the method of the present invention enables production ofhighly water-soluble biologically active compounds in nanoparticulateform. Such compounds may exhibit advantages over conventional compoundsby way of, for example, more rapid therapeutic action or lower dose. Incontrast, wet grinding techniques utilizing water (or other comparablypolar solvents) are incapable of being applied to such compounds, as theparticles dissolve appreciably in the solvent.

As will be described subsequently, selection of an appropriate grindingcompound affords particular highly advantageous applications of themethod of the present invention. Some grinding compounds appropriate foruse in the invention are readily separable from the biologically activecompound in nanoparticulate form by methods not dependent on particlesize (such methods being inappropriate due to the degradation of thegrinding compound). For example, selecting an appropriate grindingcompound that also possesses solubility properties different from thebiologically active compound in nanoparticulate form allows separationof the two by relatively straightforward selective dissolutiontechniques. Examples of such grinding compounds are provided in thedetailed description of the invention. Thus, a particularly advantageousapplication of the method of the invention is the use of a water-solublesalt as a grinding compound in conjunction with a poorly water-solublebiologically active compound.

Again, as will be described subsequently, a highly advantageous aspectof the present invention is that certain grinding compounds appropriatefor use in the method of the invention are also appropriate for use in amedicament. The present invention encompasses methods for the productionof a medicament incorporating both the biologically active compound innanoparticulate form and at least a portion of the grinding compound,medicaments so produced, and methods of treatment of an animal,including man, using a therapeutically effective amount of saidbiologically active compounds by way of said medicaments.

Analogously, as will be described subsequently, a highly advantageousaspect of the present invention is that certain grinding compoundsappropriate for use in the method of the invention are also appropriatefor use in a carrier for an agricultural chemical, such as a pesticideor a herbicide. The present invention encompasses methods for theproduction of an agricultural chemical composition incorporating boththe biologically active compound in nanoparticulate form and at least aportion of the grinding compound, and agricultural chemical compositionsso produced.

The agricultural chemical compound may include only the biologicallyactive compound in nanoparticulate form together with the milledgrinding compound or, more preferably, the biologically active compoundsin nanoparticulate form and milled grinding compound may be combinedwith one or more pharmaceutically acceptable carriers, as well as anydesired excipients or other like agents commonly used in the preparationof medicaments.

Analogously, the agricultural chemical composition may include only thebiologically active compound in nanoparticulate form together with themilled grinding compound or, more preferably, the biologically activecompounds in nanoparticulate form and milled grinding compound may becombined with one or more carriers, as well as any desired excipients orother like agents commonly used in the preparation of agriculturalchemical compositions.

In one particular form of the invention, the grinding compound is bothappropriate for use in a medicament and readily separable from thebiologically active compound in nanoparticulate form by methods notdependent on particle size. Such grinding compounds are described in thefollowing detailed description of the invention. Such grinding compoundsare highly advantageous in that they afford significant flexibility inthe extent to which the grinding compound may be incorporated with thebiologically active compound in nanoparticulate form into a medicament.

In one aspect, the invention provides novel formulations of raloxifene.Raloxifene is[6-hydroxy-2-(4-hydroxyphenyl)benzol[b]thien-3-yl][4-[2-(1-piperidinyl)ethoxy]phenyl-,and is also known as6-hydroxy-2-(4-hydrophenyl)-3-[4-(2-piperidinoethoxy)-benzoyl]benzo-[b]-thiophene.Other names for raloxifene may also be found in the literature. Thestructural formula for raloxifene is illustrated below:

This invention provides raloxifene, or a pharmaceutically acceptablesalt or solvate thereof, in particulate form having a mean particle sizeof between about 10 nm and about 500 nm.

The invention further provides methods for producing said particulateraloxifene, pharmaceutically acceptable salt or solvate thereof.

The invention also provides pharmaceutical compositions comprising orformulated using the said particulate raloxifene, or pharmaceuticallyacceptable salt or solvate thereof.

The present invention further provides the use of the said particulateraloxifene, or pharmaceutically acceptable salt or solvate thereof, inthe manufacture of a pharmaceutical composition for alleviating humanpathologies, including osteoporosis, serum lipid lowering, andinhibiting endometriosis, uterine fibrosis, and breast cancer.

The present invention further provides the use of such compositionscomprising or formulated using the said raloxifene, or pharmaceuticallyacceptable salt or solvate thereof, for alleviating human pathologies,including osteoporosis, serum lipid lowering, and inhibitingendometriosis, uterine fibrosis, and breast cancer.

In one aspect, then, the invention provides a method for producing acomposition comprising nanoparticles of a biologically active compound,the method comprising the step of:

-   -   dry milling a solid biologically active compound and a millable        grinding compound in a mill comprising a plurality of milling        bodies, for a time period sufficient to produce a solid        dispersion comprising nanoparticles of the biologically active        compound dispersed in at least partially milled grinding        compound. A pharmaceutically acceptable carrier may also be        combined with such composition to produce a pharmaceutical        composition, or a medicament.

In another aspect, the nanoparticles have an average size less than 1000nm, less than 500 nm, less than 350 nm, less than 200 nm, less than 100nm, less than 75 nm, less than 50 nm, or less than 40 nm. The particlesize of at least 50%, or 75%, of the nanoparticles may be within theaverage size range.

The time period for the milling operation is preferably between 5minutes and 8 hours, more preferably between 5 minutes and 2 hours, morepreferably between 5 minutes and 4 hours, preferably between 5 and 45minutes, more preferably between 5 and 30 minutes, most preferablybetween 10 and 25 minutes.

In another aspect of this invention, the milling medium is selected fromthe group consisting of ceramics, glasses, polymers, ferromagnetics, andmetals, such as steel balls, which may have a diameter of between 1 and20 mm, preferably between 2 and 15 mm, more preferably between 3 and 10mm.

The method of the invention is suitable for milling biologically activecompounds, such as biologics, amino acids, proteins, peptides,nucleotides, nucleic acids, and analogs homologs and first orderderivatives thereof. Many drugs are amenable to the methods of theinvention, including but not limited to diclofenac, olanzapine,sildenafil, raloxifene, and others.

In another aspect, the method further comprises the step of removing atleast a portion of the at least partially milled grinding compound.

The invention also provides a nanoparticle composition comprisingnanoparticles of a biologically active compound, formed by the processof dry milling a solid biologically active compound and a millablegrinding compound in a mill comprising a plurality of milling bodies,for a time period sufficient to produce a solid dispersion comprisingnanoparticles of the biologically active compound dispersed in at leastpartially milled grinding compound. Such nanoparticle compositions mayhave the same particle size ranges as aforementioned. Likewise, theprocess may further comprise removing at least a portion of the at leastpartially milled grinding compound.

In another aspect, the invention provides a method of treating a humanin need of such treatment comprising the step of administering to suchhuman a pharmaceutically effective amount of a nanoparticle composition,a pharmaceutical composition, or a medicament as described above.

Other aspects and advantages of the invention will become apparent tothose skilled in the art from a review of the ensuing description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that with decreasing volume percentage of diclofenac acidin NaCl grinding compound, the surface area of the diclofenacnanoparticles increases (nanoparticles after removal of grindingcompound by washing);

FIG. 2A and FIG. 2B illustrates diclofenac acid nanoparticles obtainedby dry milling a 15 vol % diclofenac acid in NaCl grinding medium, andseparated from the grinding medium by washing with 0.01 M HCl and 1 mMCTAB solution. Larger particles, as can be seen in the intensitydistribution on (b), were largely removed by centrifugation for 1 min at3,000 g to achieve a narrow size distribution of 160±30 nm, which isnumber weighted 100% (a). The amount of nanoparticles after removal ofaggregates or larger particles by centrifugation in the dispersion orsolution is greater than 80 weight %, as determined by the intensityweighted size distribution (a);

FIG. 3A and FIG. 3B comprises SEM images of olanzapine milled with NaClgrinding compound for 180 minutes, showing (a) agglomerate morphology ofolanzapine/grinding compound mixture at 10000 magnification, and (b)nanoparticulate morphology of olanzapine/grinding compound mixture at100000 magnification;

FIG. 4 comprises high resolution SEM and TEM images of washed diclofenacacid nanoparticles of 5, 10, 15, 30 and 50 wt % diclofenac acid togrinding compound ratio;

FIG. 5 is a TEM image of diclofenac acid milled with NH₄Cl and washedwith 0.1 M HCl and 1 mM CTAB, and dried on a TEM grid.;

FIG. 6 plots heat flow against temperature for diclofenac acid drymilled with NH₄Cl grinding compound, with the peak at 177° C. showingthe presence of diclofenac acid, and the peak at 194° C. being due tothe NH₄Cl grinding compound;

FIG. 7 illustrates the effect of increasing milling time of diclofenacacid with NaCl grinding compound, 15 vol %), showing that the meltingpoint shifts to lower temperatures, likely due to a decrease of thediameter of the particles of diclofenac acid;

FIG. 8A and FIG. 8B is a comparison of the dissolution profiles ofparticulate raloxifene hydrochloride of an embodiment of the inventionand commercial raloxifene hydrochloride in simulated gastric fluid andin simulated intestinal fluid;

FIGS. 9a through 9d are scanning electron micrographs comparingparticulate raloxifene hydrochloride of an embodiment of the inventionand commercial raloxifene hydrochloride;

FIG. 10 illustrates a size distribution of particulate raloxifenehydrochloride of an embodiment of the invention determined by dynamiclight scatter (DLS);

FIG. 11 compares melting points of particulate raloxifene hydrochlorideof an embodiment of the invention and commercial raloxifenehydrochloride;

FIG. 12 compares XRD-spectra for particulate raloxifene hydrochloride ofan embodiment of the invention and commercial raloxifene hydrochloride;

FIG. 13 is a solution ¹H-NMR spectrum for particulate raloxifenehydrochloride of an embodiment of the invention;

FIG. 14 compares the FT-IR spectra of particulate raloxifenehydrochloride of an embodiment of the invention with commercialraloxifene hydrochloride;

FIG. 15 compares XRD spectra of raloxifene hydrochloride at variousstages of processing according to a method of the present invention;

FIG. 16 is a scanning electron micrograph of particulate raloxifenehydrochloride according to an embodiment of the invention;

FIG. 17 compares FT-IR spectra of raloxifene hydrochloride at variousstages of processing according to an embodiment of the method of thepresent invention;

FIG. 18A through 18D is a scanning electron micrograph of raloxifene(free base) as obtained (a and b) and after processing by milling withsodium chloride (c and d).

FIG. 19 shows the structures of ionic surfactants utilized in someembodiments of the method of the invention;

FIG. 20A and FIG. 20B is a scanning electron micrograph of particulateraloxifene (free base) according to an embodiment of the invention;

FIG. 21 compares XRD spectra of raloxifene (free base) at various stagesof processing according to a method of the present invention;

FIG. 22 compares FT-IR spectra of raloxifene hydrochloride at variousstages of processing according to an embodiment of the method of thepresent invention;

FIG. 23A through 23D provides concentration v time data for animalexperiments comparing particulate raloxifene hydrochloride of anembodiment of the invention and commercial raloxifene hydrochloride;

FIG. 24A and FIG. 24B provides the data of FIG. 16 in graphical andtabular form;

FIG. 25 provides mean pharmacokinetic data in tabular form; and

FIG. 26A through 26C provides an additional comparison of C_(max) andAUC_(0-t) results.

FIG. 27 comprises high resolution SEM images showing washed particulatefenofibrate produced by milling in an attrition mill for 30, 45 and 60minutes.

FIG. 28A and FIG. 28B comprises a high resolution SEM micrograph ofraloxifene HCl in a lactose grinding compound;

FIG. 29 compares in vitro dissolution of raloxifene HCl API withraloxifene milled with both sodium chloride and lactose as grindingcompound and without removal of the grinding compound; and

FIG. 30A and FIG. 30B comprises SEM micrographs showing that olanzapinefree base can be ground with lactose to a fine powder with some largeragglomerates (FIG. 30a ) and very fine particles of about 50-100 nm(FIG. 30b ).

DETAILED DESCRIPTION OF THE INVENTION General

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in the specification, individually or collectively andany and all combinations or any two or more of the steps or features.

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended for the purpose ofexemplification only. Functionally equivalent products, compositions andmethods are clearly within the scope of the invention as describedherein.

The invention described herein may include one or more ranges of values(e.g. size, concentration etc). A range of values will be understood toinclude all values within the range, including the values defining therange, and values adjacent to the range that lead to the same orsubstantially the same outcome as the values immediately adjacent tothat value which defines the boundary to the range.

The entire disclosures of all publications (including patents, patentapplications, journal articles, laboratory manuals, books, or otherdocuments) cited herein are hereby incorporated by reference. Inclusiondoes not constitute an admission is made that any of the referencesconstitute prior art or are part of the common general knowledge ofthose working in the field to which this invention relates.

Throughout this specification, unless the context requires otherwise,the word “comprise” or variations, such as “comprises” or “comprising”will be understood to imply the inclusion of a stated integer, or groupof integers, but not the exclusion of any other integers or group ofintegers. It is also noted that in this disclosure, and particularly inthe claims and/or paragraphs, terms such as “comprises”, “comprised”,“comprising” and the like can have the meaning attributed to it in USPatent law; e.g., they can mean “includes”, “included”, “including”, andthe like.

“Therapeutically effective amount” as used herein with respect tomethods of treatment and in particular drug dosage, shall mean thatdosage that provides the specific pharmacological response for which thedrug is administered in a significant number of subjects in need of suchtreatment. It is emphasized that “therapeutically effective amount,”administered to a particular subject in a particular instance will notalways be effective in treating the diseases described herein, eventhough such dosage is deemed a “therapeutically effective amount” bythose skilled in the art. It is to be further understood that drugdosages are, in particular instances, measured as oral dosages, or withreference to drug levels as measured in blood.

The term “inhibit” is defined to include its generally accepted meaningwhich includes prohibiting, preventing, restraining, and lowering,stopping, or reversing progression or severity, and such action on aresultant symptom. As such the present invention includes both medicaltherapeutic and prophylactic administration, as appropriate.

The term “mean particle size” is defined as equivalent sphericaldiameter as determined by laser light diffraction scattering.

Because the particles in the raw state as well as after milling or otherparticle size reduction techniques are irregular in shape, it isnecessary to characterize them not by measurement of an actual size suchas thickness or length, but by measurement of a property of theparticles which is related to the sample property possessed by atheoretical spherical particle. The particles are thus allocated an“equivalent spherical diameter”.

The values found from characterizing a large number of “unknown”particles can be plotted frequency vs. diameter or in other methodsweight vs. diameter, usually adopting percentage undersize values forfrequency or weight. This gives a characteristic curve representing sizedistribution of the sample, i.e., cumulative percentage undersizedistribution curve. Values from this can be read off directly or plottedon log-probability paper to give an appropriate straight line. The meanequivalent spherical volume diameter is the 50% undersize value.

Methods of determining particle sizes are known in the art, and themethod by which the particle sizes of the raloxifene hydrochloride ofthe present invention are measured is described herein. However, othermethods may be employed—see, for example, the methods described in U.S.Pat. No. 4,605,517 (Riley et al.).

As used herein, particle size refers to a number average particle sizeas measured by conventional particle size measuring techniques wellknown to those skilled in the art, such as sedimentation field flowfractionation, photon correlation spectroscopy, or disk centrifugation.By “an effective average particle size of less than about 400 nm” it ismeant that at least 90% of the particles have a number average particlesize of less than about 400 nm when measured by the above-notedtechniques.

As used herein, the term “effective mean particle diameter” is definedas the mean diameter of the smallest circular hole through which aparticle can pass freely. For example, the effective mean particlediameter of a spherical particle corresponds to the mean particlediameter and the effective mean particle diameter of an ellipsoidalparticle corresponds to the mean length of the longest minor axis.

Throughout this specification, unless the context requires otherwise,the term “solvate” is used to describe an aggregate that comprises oneor more molecules of the solute, such as raloxifene, with one or moremolecules of solvent.

Throughout this specification, unless the context requires otherwise,the term “pharmaceutically acceptable salt” refers to either acid orbase addition salts which are known to be non-toxic and are commonlyused in the pharmaceutical literature. The pharmaceutically acceptablesalts generally have enhanced solubility characteristics compared to thecompound from which they are derived, and thus are often more amenableto formulation as liquids or emulsions. The compounds used in themethods of this invention primarily form pharmaceutically acceptableacid addition salts with a wide variety of organic and inorganic acids,and include the physiologically acceptable salts which are often used inpharmaceutical chemistry. Such salts are also part of this invention.

The pharmaceutically acceptable acid addition salts are typically formedby reacting raloxifene with an equimolar or excess amount of acid. Thereactants are generally combined in a mutual solvent such as diethylether or ethyl acetate. The salt normally precipitates out of solutionwithin about one hour to 10 days and can be isolated by filtration, orthe solvent can be stripped off by conventional means.

Throughout this specification, unless the context requires otherwise,the phrase “dry mill” or variations, such as “dry milling”, should beunderstood to refer to milling in at least the substantial absence ofliquids. If liquids are present, they are present in such amounts thatthe contents of the mill retain the characteristics of a paste or,preferably, a dry powder.

“Flowable” means a powder having physical characteristics rendering itsuitable for an automatic or semi-automatic manufacturing process as,for example, would be used for the manufacture of pharmaceuticalcompositions and formulations.

Other definitions for selected terms used herein may be found within thedetailed description of the invention and apply throughout. Unlessotherwise defined, all other scientific and technical terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which the invention belongs.

Throughout this specification, unless the context requires otherwise,the term “nanoparticulate form” includes nanoparticle compositions,wherein the composition comprises at least nanoparticles having anaverage particle size smaller than 1000 nm.

“Conventional” in the context of the form of biologically activecompounds, agents or drugs refers to non-nanoparticulate compositions.Non-nanoparticulate active agents have an effective average particlesize of greater than about 2 microns, meaning that at least 50% of theactive agent particles have a size greater than about 2 microns.

A “solid solution” consists of one phase only, irrespective of thenumber of differing components present. A solid solution may beclassified as continuous, discontinuous, substitutional, interstitial oramorphous. Typical solid solutions have a crystalline structure, inwhich the solute molecules can either substitute for solvent moleculesin the crystal lattice or fit into the interstices between the solventmolecules. Interstitial crystalline solid solutions occur when thedissolved molecules occupy the interstitial spaces between the solventmolecules in the crystal lattice. Amorphous solid solutions occur whenthe solute molecules are dispersed molecularly but irregularly withinthe amorphous solvent.

The term “a solid dispersion” in general means a system in solid statecomprising at least two components, wherein one component is dispersedmore or less evenly throughout the other component or components.

Other definitions for selected terms used herein may be found within thedetailed description of the invention and apply throughout. Unlessotherwise defined, all other scientific and technical terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which the invention belongs.

Specific

In one embodiment, the present invention is directed to a method forproducing for the preparation of a biologically active compound innanoparticulate form, the method comprising the step of:

-   -   dry milling a mixture of a solid biologically active compound        and a millable grinding compound, in a mill comprising a        plurality of milling bodies, to produce a solid dispersion or        solution comprising nanoparticulate biologically active compound        dispersed in at least partially milled grinding compound.

The solid dispersion or solution may then be separated from the millingbodies and removed from the mill.

In one aspect, the grinding compound is separated from the dispersion orsolution. In one aspect, where the grinding compound is not fullymilled, the unmilled grinding compound is separated from thenanoparticulate biologically active compound. In a further aspect, atleast a portion of the milled grinding compound is separated from thenanoparticulate biologically active compound.

The milling bodies are essentially resistant to fracture and erosion inthe dry milling process. The quantity of the grinding compound relativeto the quantity of biologically active compound in nanoparticulate form,and the extent of milling of the grinding compound, is sufficient toinhibit reagglomeration of the biologically active compound innanoparticulate form.

The grinding compound is neither chemically nor mechanically reactivewith the pharmaceutical compound under the conditions present in theprocess of the invention.

The present invention also relates to biologically active compounds innanoparticulate form produced by said methods, to medicaments producedusing said biologically active compounds in nanoparticulate form and tomethods of treatment of an animal, including man, using atherapeutically effective amount of said biologically active compoundsadministered by way of said medicaments.

Grinding Compound

As stated above, the method of the present invention requires thegrinding compound to be milled with the pharmaceutical compound; thatis, the grinding compound will physically degrade under the dry millingconditions of the invention to facilitate the formation and retention ofthe biologically active compound in nanoparticulate form. The preciseextent of degradation required will depend on certain properties of thegrinding compound and the biologically active compound (for example, anycharge distribution or surface effects causing the grinding compound tohave a greater or lesser affinity for the biologically active compound),the ratio of biologically active compound to grinding compound, and thedesired particle size and particle size distribution of thenanoparticles comprising the biologically active compound innanoparticulate form.

In one embodiment of the invention, the milled grinding compound is of acomparable particle size to the nanoparticulate biologically activecompound.

The physical properties of the grinding compound necessary to achievethe requisite degradation are dependant on the precise millingconditions. For example a harder grinding compound may degrade to asufficient extent provided under more vigorous dry milling conditions.

Physical properties of the grinding compound relevant to the extent thatthe agent will degrade under dry milling conditions include hardness,friability, as measured by indicia such as fracture toughness andbrittleness index.

A low hardness (typically a Mohs Hardness less than 7) of thebiologically active compound is desirable to ensure fracture of theparticles during processing, so that nanocomposite microstructuresdevelop during milling.

Preferably, the grinding compound is of low abrasivity. Low abrasivityis desirable to minimise contamination of the dispersion or solution ofthe biologically active compound in nanoparticulate form in the grindingcompound by the milling bodies and/or the milling chamber of the mediamill. An indirect indication of the abrasivity can be obtained bymeasuring the level of milling-based contaminants.

Preferably, the grinding compound has a low tendency to agglomerateduring dry milling. While it is difficult to objectively quantify thetendency to agglomerate during milling, it is possible to obtain asubjective measure by observing the level of “caking” of the grindingcompound on the milling bodies and the milling chamber of the media millas dry milling progresses.

The grinding compound may be an inorganic or organic compound. In oneembodiment, the grinding compound is selected from the following: sodiumhydrogen sulfate, sodium hydrogen carbonate, sodium hydroxide, orsuccinic acid; crystalline organic acids, for example (but not limitedto) fumaric acid, maleic acid, tartaric acid, citric acid);alternatively ammonium salts (or salts of volatile amines), for example(but not limited to) ammonium chloride, methylamine hydrochloride,ammonium bromide, crystalline hydroxides, hydrogen carbonates, hydrogencarbonates of pharmaceutical acceptable alkali metals, such as but notlimited by, sodium, potassium, lithium, calcium, and barium, sodiumsulphate, sodium chloride, sodium metabisulphite, sodium thiosulphate,ammonium chloride, Glauber's salt, ammonium carbonate, sodiumbisulphate, magnesium sulphate, potash alum, potassium chloride, sodiumcarbonate, sodium bicarbonate, potassium carbonate, potassiumbicarbonate.

In a preferred embodiment, the grinding compound is a compound that isconsidered GRAS (generally regarded as safe) by persons skilled in thepharmaceutical arts.

Relative Quantity of Grinding Compound

The quantity of the grinding compound relative to the quantity ofbiologically active compound in nanoparticulate form and the extent ofdegradation of the grinding compound, determine whether reagglomerationof the biologically active compound in nanoparticulate form is at leastinhibited.

Further, the extent of degradation of the grinding compound under thedry milling conditions of the invention may affect the quantity ofgrinding compound required to produce the biologically active compoundin nanoparticulate form, such that grinding compounds that degrade to agreater extent are required in smaller relative quantities.

For example, it may be possible for the volume fraction of thenanoparticles comprising the biologically active compound innanoparticulate form to be greater than the theoretical percolationthreshold, which, for 3-dimensional random dispersions of sphericalparticles, is around 15 vol %. In a preferred form of the invention, thevolume fraction of the nanoparticles comprising the biologically activecompound in nanoparticulate form is less than about 25 vol %. Morepreferably, the volume fraction of the nanoparticles comprising thebiologically active compound in nanoparticulate form is less than about20 vol %. In a highly preferred form of the invention, the volumefraction of the nanoparticles comprising the biologically activecompound in nanoparticulate form is less than about 15 vol %.

Milling Bodies

In the method of the present invention, the milling bodies arepreferably chemically inert and rigid. The term “chemically-inert”, asused herein, means that the milling bodies do not react chemically withthe biologically active compound or the grinding compound.

As described above, the milling bodies are essentially resistant tofracture and erosion in the milling process.

The milling bodies are desirably provided in the form of bodies whichmay have any of a variety of smooth, regular shapes, flat or curvedsurfaces, and lacking sharp or raised edges. For example, suitablemilling bodies can be in the form of bodies having ellipsoidal, ovoid,spherical or right cylindrical shapes. Preferably, the milling bodiesare provided in the form of one or more of beads, balls, spheres, rods,right cylinders, drums or radius-end right cylinders (i.e., rightcylinders having hemispherical bases with the same radius as thecylinder).

Depending on the nature of the biologically active compound substrateand the grinding compound, the milling media bodies desirably have aneffective mean particle diameter (i.e. “particle size”) between about0.1 and 30 mm, more preferably between about 1 and about 15 mm, stillmore preferably between about 3 and 10 mm.

The milling bodies may comprise various materials such as ceramic,glass, metal or polymeric compositions, in a particulate form. Suitablemetal milling bodies are typically spherical and generally have goodhardness (i.e. RHC 60-70), roundness, high wear resistance, and narrowsize distribution and can include, for example, balls fabricated fromtype 52100 chrome steel, type 316 or 440C stainless steel or type 1065high carbon steel.

Preferred ceramic materials, for example, can be selected from a widearray of ceramics desirably having sufficient hardness and resistance tofracture to enable them to avoid being chipped or crushed during millingand also having sufficiently high density. Suitable densities formilling media can range from about 1 to 15 g/cm³. Preferred ceramicmaterials can be selected from steatite, aluminum oxide, zirconiumoxide, zirconia-silica, yttria-stabilized zirconium oxide,magnesia-stabilized zirconium oxide, silicon nitride, silicon carbide,cobalt-stabilized tungsten carbide, and the like, as well as mixturesthereof.

Preferred glass milling media are spherical (e.g. beads), have a narrowsize distribution, are durable, and include, for example, lead-free sodalime glass and borosilicate glass. Polymeric milling media arepreferably substantially spherical and can be selected from a wide arrayof polymeric resins having sufficient hardness and friability to enablethem to avoid being chipped or crushed during milling,abrasion-resistance to minimize attrition resulting in contamination ofthe product, and freedom from impurities such as metals, solvents, andresidual monomers.

Preferred polymeric resins, for example, can be selected fromcrosslinked polystyrenes, such as polystyrene crosslinked withdivinylbenzene, styrene copolymers, polyacrylates such aspolymethylmethacrylate, polycarbonates, polyacetals, vinyl chloridepolymers and copolymers, polyurethanes, polyamides, high densitypolyethylenes, polypropylenes, and the like. The use of polymericmilling media to grind materials down to a very small particle size (asopposed to mechanochemical synthesis) is disclosed, for example, in U.S.Pat. Nos. 5,478,705 and 5,500,331. Polymeric resins typically can havedensities ranging from about 0.8 to 3.0 g/cm³. Higher density polymericresins are preferred. Alternatively, the milling media can be compositeparticles comprising dense core particles having a polymeric resinadhered thereon. Core particles can be selected from materials known tobe useful as milling media, for example, glass, alumina, zirconiasilica, zirconium oxide, stainless steel, and the like. Preferred corematerials have densities greater than about 2.5 g/cm³.

In one embodiment of the invention, the milling media are formed from aferromagnetic material, thereby facilitating removal of contaminantsarising from wear of the milling media by the use of magnetic separationtechniques.

Each type of milling body has its own advantages. For example, metalshave the highest specific gravities, which increase grinding efficiencydue to increased impact energy. Metal costs range from low to high, butmetal contamination of final product can be an issue. Glasses areadvantageous from the standpoint of low cost and the availability ofsmall bead sizes as low as 0.004 mm. However, the specific gravity ofglasses is lower than other media and significantly more milling time isrequired. Finally, ceramics are advantageous from the standpoint of lowwear and contamination, ease of cleaning, and high hardness.

Dry Milling

In the dry milling process of the present invention, the biologicallyactive compound substrate and grinding compound, in the form ofcrystals, powders, or the like, are combined in suitable proportionswith the plurality of milling bodies in a milling chamber that ismechanically agitated (i.e., with or without stirring) for apredetermined period of time at a predetermined intensity of agitation.Typically, a milling apparatus is used to impart motion to the millingbodies by the external application of agitation, whereby varioustranslational, rotational or inversion motions or combinations thereofare applied to the milling chamber and its contents, or by the internalapplication of agitation through a rotating shaft terminating in ablade, propeller, impeller or paddle or by a combination of bothactions.

During milling, motion imparted to the milling bodies can result inapplication of shearing forces as well as multiple impacts or collisionshaving significant intensity between milling bodies and particles of thereactant powders. The nature and intensity of the forces applied by themilling bodies to the biologically active compound and the grindingcompound is influenced by a wide variety of processing parametersincluding: the type of milling apparatus; the intensity of the forcesgenerated, the kinematic aspects of the process; the size, density,shape, and composition of the milling bodies; the weight ratio of thebiologically active compound and grinding compound mixture to themilling bodies; the duration of milling; the physical properties of boththe biologically active compound and the grinding compound; theatmosphere present during activation; and others.

Advantageously, the media mill is capable of repeatedly or continuouslyapplying mechanical compressive forces and shear stress to thebiologically active compound substrate and the grinding compound.Suitable media mills include but are not limited to the following:high-energy ball, sand, bead or pearl mills, basket mill, planetarymill, vibratory action ball mill, multi-axial shaker/mixer, stirred ballmill, horizontal small media mill, multi-ring pulverizing mill, and thelike, including small milling media. The milling apparatus also cancontain one or more rotating shafts.

In a preferred form of the invention, the dry milling is effected a ballmill. Throughout the remainder of the specification reference will bemade to dry milling being carried out by way of a ball mill. Examples ofthis type of mill are attritor mills, nutating mills, tower mills,planetary mills, vibratory mills and gravity-dependent-type ball mills.It will be appreciated that dry milling in accordance with the method ofthe invention may also be achieved by any suitable means other than ballmilling. For example, dry milling may also be achieved using jet mills,rod mills, roller mills or crusher mills.

Biologically Active Compound

The biologically active compound includes therapeutically activecompounds, including compounds for veterinary and human use, andagricultural compounds such as pesticides, herbicides and fungicides,germinating agents and the like.

In a preferred form of the invention, the biologically active compoundis an organic compound. In a highly preferred form of the invention, thebiologically active compound is an organic, therapeutically activecompounds for veterinary or human use. In a highly preferred form of theinvention, the biologically active compound is an organic,therapeutically active compounds for human use.

The biologically active compound substrate is ordinarily a compound forwhich one of skill in the art desires improved properties arising fromsmaller particle sizes. The biologically active compound substrate maybe a conventional active agent or drug, although the process of theinvention may be employed on formulations or agents that already havereduced particle size compared to their conventional form.

Biologically active compounds suitable for use in the invention includebiologics, amino acids, proteins, peptides, nucleotides, nucleic acids,and analogs, homologs and first order derivatives thereof. Thebiologically active compound can be selected from a variety of knownclasses of drugs, including, but not limited to: anti-obesity drugs,central nervous system stimulants, carotenoids, corticosteroids,elastase inhibitors, anti-fungals, oncology therapies, anti-emetics,analgesics, cardiovascular agents, anti-inflammatory agents, such asNSAIDs and COX-2 inhibitors, anthelmintics, anti-arrhythmic agents,antibiotics (including penicillins), anticoagulants, antidepressants,antidiabetic agents, antiepileptics, antihistamines, antihypertensiveagents, antimuscarinic agents, antimycobacterial agents, antineoplasticagents, immunosuppressants, antithyroid agents, antiviral agents,anxiolytics, sedatives (hypnotics and neuroleptics), astringents,alpha-adrenergic receptor blocking agents, beta-adrenoceptor blockingagents, blood products and substitutes, cardiac inotropic agents,contrast media, cough suppressants (expectorants and mucolytics),diagnostic agents, diagnostic imaging agents, diuretics, dopaminergics(anti-Parkinsonian agents), haemostatics, immunological agents, lipidregulating agents, muscle relaxants, parasympathomimetics, parathyroidcalcitonin and biphosphonates, prostaglandins, radio-pharmaceuticals,sex hormones (including steroids), anti-allergic agents, stimulants andanoretics, sympathomimetics, thyroid agents, vasodilators, andxanthines.

A description of these classes of active agents and a listing of specieswithin each class can be found in Martindale's The Extra Pharmacopoeia,31st Edition (The Pharmaceutical Press, London, 1996), specificallyincorporated by reference. Another source of active agents is thePhysicians Desk Reference (60th Ed., pub. 2005), familiar to those ofskill in the art. The active agents are commercially available and/orcan be prepared by techniques known in the art.

An exhaustive list of drugs for which the methods of the invention aresuitable would be burdensomely long for this specification; however,reference to the general pharmacopoeia listed above would allow one ofskill in the art to select virtually any drug to which the method of theinvention may be applied.

Notwithstanding the general applicability of the method of theinvention, more specific examples of biologically active compoundsinclude, but are not limited to: haloperidol (dopamine antagonist), DLisoproterenol hydrochloride (β-adrenergic agonist), terfenadine(H1-antagonist), propranolol hydrochloride (β-adrenergic antagonist),desipramine hydrochloride (antidepressant), salmeterol (b2-selectiveadrenergic agonist), sildenafil citrate, tadalafil and vardenafil. Minoranalgesics (cyclooxygenase inhibitors), fenamic acids, Piroxicam, Cox-2inhibitors, and Naproxen, and others, may all benefit from beingprepared in a nanoparticulate form.

As discussed in the context of the background to the invention,biologically active compounds that are poorly water soluble atphysiological pH will particularly benefit from being prepared innanoparticulate form, and the method of the present invention isparticularly advantageously applied to compounds that are poorly watersoluble at physiological pH.

Such compounds include, but are not limited to: albendazole, albendazolesulfoxide, alfaxalone, acetyl digoxin, acyclovir analogs, alprostadil,aminofostin, anipamil, antithrombin III, atenolol, azidothymidine,beclobrate, beclomethasone, belomycin, benzocaine and derivatives, betacarotene, beta endorphin, beta interferon, bezafibrate, binovum,biperiden, bromazepam, bromocryptine, bucindolol, buflomedil,bupivacaine, busulfan, cadralazine, camptothesin, canthaxanthin,captopril, carbamazepine, carboprost, cefalexin, cefalotin, cefamandole,cefazedone, cefluoroxime, cefinenoxime, cefoperazone, cefotaxime,cefoxitin, cefsulodin, ceftizoxime, chlorambucil, chromoglycinic acid,ciclonicate, ciglitazone, clonidine, cortexolone, corticosterone,cortisol, cortisone, cyclophosphamide, cyclosporin A and othercyclosporins, cytarabine, desocryptin, desogestrel, dexamethasone esterssuch as the acetate, dezocine, diazepam, diclofenac, dideoxyadenosine,dideoxyinosine, digitoxin, digoxin, dihydroergotamine, dihydroergotoxin,diltiazem, dopamine antagonists, doxorubicin, econazole, endralazine,enkephalin, enalapril, epoprostenol, estradiol, estramustine,etofibrate, etoposide, factor ix, factor viii, felbamate, fenbendazole,fenofibrate, fexofenedine, flunarizin, flurbiprofen, 5-fluorouracil,flurazepam, fosfomycin, fosmidomycin, furosemide, gallopamil, gammainterferon, gentamicin, gepefrine, gliclazide, glipizide, griseofulvin,haptoglobulin, hepatitis B vaccine, hydralazine, hydrochlorothiazide,hydrocortisone, ibuprofen, ibuproxam, indinavir, indomethacin, iodinatedaromatic x-ray contrast agents such as iodamide, ipratropium bromide,ketoconazole, ketoprofen, ketotifen, ketotifen fumarate, K-strophanthin,labetalol, lactobacillus vaccine, lidocaine, lidoflazin, lisuride,lisuride hydrogen maleate, lorazepam, lovastatin, mefenamic acid,melphalan, memantin, mesulergin, metergoline, methotrexate, methyldigoxin, methylprednisolone, metronidazole, metisoprenol, metipranolol,metkephamide, metolazone, metoprolol, metoprolol tartrate, miconazole,miconazole nitrate, minoxidil, misonidazol, molsidomin, nadolol,nafiverine, nafazatrom, naproxen, natural insulins, nesapidil,nicardipine, nicorandil, nifedipine, niludipin, nimodipine, nitrazepam,nitrendipine, nitrocamptothesin, 9-nitrocamptothesin, olanzapine,oxazepam, oxprenolol, oxytetracycline, penicillins such as penicillin Gbenethamine, penecillin O, phenylbutazone, picotamide, pindolol,piposulfan, piretanide, piribedil, piroxicam, pirprofen, plasminogeniciactivator, prednisolone, prednisone, pregnenolone, procarbacin,procaterol, progesterone, proinsulin, propafenone, propanolol,propentofyllin, propofol, propranolol, raloxifene, rifapentin,simvastatin, semi-synthetic insulins, sobrerol, somastotine and itsderivatives, somatropin, stilamine, sulfinalol hydrochloride,sulfinpyrazone, suloctidil, suprofen, sulproston, synthetic insulins,talinolol, taxol, taxotere, testosterone, testosterone propionate,testosterone undecanoate, tetracane HI, tiaramide HCl, tolmetin,tranilast, triquilar, tromantadine HCl, urokinase, valium, verapamil,vidarabine, vidarabine phosphate sodium salt, vinblastine, vinburin,vincamine, vincristine, vindesine, vinpocetine, vitamin A, vitamin Esuccinate, and x-ray contrast agents. Drugs can be neutral species orbasic or acidic as well as salts such as exist in the presence of anaqueous buffer.

In addition, some biologically active compounds may have the benefit ofabsorption through the skin if presented in a nanoparticle formulation.Such biologically active compounds include, but are not limited to,Voltaren (diclofenac), rofecoxib, and ibuprofen.

Conveniently, the biologically active compound is capable ofwithstanding temperatures that are typical in uncooled dry milling,which may exceed 80° C. Therefore, compounds with a melting point about80° C. or greater are suitable. For biologically active compounds withlower melting points, the media mill may be cooled, thereby allowingcompounds with significantly lower melting temperatures to be processedaccording to the method of the invention. For instance, a simplewater-cooled mill will keep temperatures below 50° C., or chilled watercould be used to further lower the milling temperature. Those skilled inthe art will understand that a reaction mill could be designed to run atany temperature between say −190 to 500° C. For some biologically activecompounds it may be advantageous to control the milling temperature totemperatures significantly below the melting points of the biologicallyactive compounds.

The biologically active compound substrate is obtained in a conventionalform commercially and/or prepared by techniques known in the art.

It is preferred, but not essential, that the particle size of thebiologically active compound substrate be less than about 100 μm, asdetermined by sieve analysis. If the coarse particle size of thebiologically active compound substrate is greater than about 100 μm,then it is preferred that the particles of the biologically activecompound substrate be reduced in size to less than 100 μm using aconventional milling method such as airjet or fragmentation milling.

Biologically Active Compound in Nanoparticulate Form

Preferably, the biologically active compound in nanoparticulate formcomprises nanoparticles of biologically active compound of an averageparticle size diameter less than 1000 nm, preferably less than 500 nm,preferably less than 350 nm, preferably less than 200 nm, preferablyless than 100 nm, preferably less than 75 nm, more preferably less than50 nm, and in some cases less than 30 nm.

Preferably, the biologically active compound in nanoparticulate formcomprises nanoparticles of biologically active compound of between about1 nm to about 200 nm, or more preferably between about 5 nm to about 100nm, more preferably between about 5 and 50 nm, more preferably stillbetween about 10 nm to about 40 nm. In a highly preferred embodiment ofthe invention, the nanoparticles of biologically active compound arebetween about 20 nm and 30 nm in size. These sizes refer tonanoparticles either fully dispersed or partially agglomerated. Forexample, where two 20 nm particles agglomerate, the resulting entity isa nanoparticle about 40 nm in size and thus would still be considered ananoparticle within the meaning of the invention. Stated alternatively,the nanoparticles of biologically active compound will preferably havean average size less than 200 nm, more preferably less than 100 nm, morepreferably less than 75 nm, more preferably less than 50 nm, and morepreferably less than 40 nm, where the average size refers tonanoparticles either fully dispersed or partially agglomerated asdescribed above.

Preferably, the nanoparticles of the biologically active compound innanoparticulate form are distributed in size so that at least 50% of thenanoparticles have a size within the average range, more preferably atleast 60%, more preferably at least 70%, and still more preferably atleast 75% of the nanoparticles have a size within the average range.

Agglomerates

Agglomerates comprising particles of biologically active compound innanoparticulate form, said particles having a mean particle size withinthe ranges specified above, should be understood to fall within thescope of the present invention, regardless of whether the agglomeratesexceed 1000 nm in size.

Time

Preferably, the biologically active compound substrate and the grindingcompound are dry milled for the shortest time necessary to form thesolid dispersion or solution of the biologically active compound innanoparticulate form in the grinding compound to minimise any possiblecontamination from the media mill and/or the plurality of millingbodies. This time varies greatly, depending on the biologically activecompound and the grinding compound, and may range from as short as 5minutes to several hours. Dry milling times in excess of 2 hours maylead to degradation of the biologically active compound innanoparticulate form and an increased level of undesirable contaminants.

Suitable rates of agitation and total milling times are adjusted for thetype and size of milling apparatus as well as the milling media, theweight ratio of the substrate biologically active compound and grindingcompound mixture to the plurality of milling bodies, the chemical andphysical properties of the substrate biologically active compound andgrinding compound, and other parameters that may be optimizedempirically.

The time may range from between 5 minutes and 2 hours, 5 minutes and 1hour, 5 minutes and 45 minutes, 5 minutes and 30 minutes, and 10 minutesand 20 minutes.

Separation of the Grinding Compound from the Biologically ActiveCompound in Nanoparticulate Form

In one embodiment, the method further comprises the step of;

-   -   Separating at least a portion of the milled grinding compound        from the biologically active compound in nanoparticulate form.

Any portion of the grinding compound may be removed, including but notlimited to 10%, 25%, 50%, 75%, or substantially all of the grindingcompound.

In some embodiments of the invention, a significant portion of themilled grinding compound may comprise particles of a size similar toand/or smaller than the particles comprising the biologically activecompound in nanoparticulate form. Where the portion of the milledgrinding compound to be separated from the particles comprising thebiologically active compound in nanoparticulate form comprises particlesof a size similar to and/or smaller than the particles comprising thebiologically active compound in nanoparticulate form, separationtechniques based on size distribution are inapplicable.

In these circumstances, the method of the present invention may involveseparation of at least a portion of the milled grinding compound fromthe biologically active compound in nanoparticulate form by techniquesincluding but not limited to electrostatic separation, magneticseparation, centrifugation (density separation), hydrodynamicseparation, froth flotation.

Advantageously, the step of removing at least a portion of the milledgrinding compound from the biologically active compound innanoparticulate form may be performed through means such as selectivedissolution, washing, or sublimation.

In one form of the invention, the grinding compound possesses solubilityproperties in a solvent different from the biologically active compoundin nanoparticulate form and the step of removing at least a portion ofthe grinding compound from the biologically active compound innanoparticulate form is performed by washing the solid dispersion orsolution of the biologically active compound in nanoparticulate form inthe grinding compound with the solvent.

Appropriate solvents may be acid, alkaline or neutral aqueous solutions,or an organic solvent. This may be any solvent in which the drug isinsoluble but the matrix is soluble or alternatively in which thebiologically active compound in nanoparticulate form and grindingcompound may be separated by differential centrifugation.

As described above, appropriate grinding compound include a number ofhighly water soluble inorganic salts. Where the biologically activecompound is poorly water soluble, a particularly appropriate grindingcompound is thus a water soluble salt as this facilitates facileseparation of the grinding compound from the biologically activecompound in nanoparticulate form by contacting the solid solution ordispersion of the biologically active compound in nanoparticulate formin the grinding compound with water.

Examples of poorly water-soluble biologically active compounds areprovided above.

Examples of water soluble inorganic salts include: sodium sulphate,sodium chloride, sodium metabisulphite, sodium thiosulphate, ammoniumchloride, Glauber's salt, ammonium carbonate, sodium bisulphate,magnesium sulphate, potash alum, potassium chloride, sodium carbonate,sodium bicarbonate, potassium carbonate, potassium bicarbonate.

Preferred water soluble inorganic salts include sodium chloride,ammonium chloride, potash alum, potassium chloride, potassium bromideand sodium sulphate, especially anhydrous sodium sulphate.

In a highly convenient form of the invention, the grinding compound issodium chloride. The sodium chloride may be provided in dendritic,granular or ordinary cubic form.

In some cases, the biologically active compound in nanoparticulate formresulting from at least partial removal of the grinding compound mayrequire stabilization with a surface stabilizer. Example surfacestabilizers include CTAB, cetyl pyridinium chloride, gelatin, casein,phosphatides, dextran, glycerol, gum acacia, cholesterol, tragacanth,stearic acid, stearic acid esters and salts, calcium stearate, glycerolmonostearate, cetostearyl alcohol, cetomacrogol emulsifying wax,sorbitan esters, polyoxyethylene alkyl ethers, polyoxyethylene castoroil derivatives, polyoxyethylene sorbitan fatty acid esters,polyethylene glycols, dodecyl trimethyl ammonium bromide,polyoxyethylene stearates, colloidal silicon dioxide, phosphates, sodiumdodecylsulfate, carboxymethylcellulose calcium, hydroxypropylcelluloses, hydroxypropyl methylcellulose, carboxymethylcellulosesodium, methylcellulose, hydroxyethylcellulose,hydroxypropylmethyl-cellulose phthalate, noncrystalline cellulose,magnesium aluminum silicate, triethanolamine, polyvinyl alcohol,polyvinylpyrrolidone, 4-(1,1,3,3-tetramethylbutyl)-phenol polymer withethylene oxide and formaldehyde, poloxamers, poloxamines, a chargedphospholipid, dimyristoyl phophatidyl glycerol, dioctylsulfosuccinate,dialkylesters of sodium sulfosuccinic acid, dioctyl sodiumsulfosuccinate, sodium lauryl sulfate, alkyl aryl polyether sulfonates,mixtures of sucrose stearate and sucrose distearate, triblock copolymersof the structure: -(-PEO)-(-PBO-)-(-PEO-)-,p-isononylphenoxypoly-(glycidol), decanoyl-N-methylglucamide; n-decylβ-D-glucopyranoside, n-decyl β-D-maltopyranoside, n-dodecylβ-D-glucopyranoside, n-dodecyl β-D-maltoside,heptanoyl-N-methylglucamide, n-heptyl-β-D-glucopyranoside, n-heptylβ-D-thioglucoside, n-hexyl β-D-glucopyranoside,nonanoyl-N-methylglucamide, n-noyl β-D-glucopyranoside,octanoyl-N-methylglucamide, n-octyl-β-D-glucopyranoside, octylβ-D-thioglucopyranoside, lysozyme, a PEG derivatized phospholipid, PEGderivatized cholesterol, a PEG derivatized cholesterol derivative, PEGderivatized vitamin A, PEG derivatized vitamin E, and random copolymersof vinyl acetate and vinyl pyrrolidone, and/or mixtures of any of theforegoing. Facilitating agents may also include at least one cationicsurface stabilizer selected from the group consisting of a polymer, abiopolymer, a polysaccharide, a cellulosic, an alginate, a nonpolymericcompound, and a phospholipid. Facilitating agents may also include atleast one surface stabilizer selected from the group consisting ofcationic lipids, benzalkonium chloride, sulfonium compounds, phosphoniumcompounds, quarternary ammonium compounds,benzyl-di(2-chloroethyl)ethylammonium bromide, coconut trimethylammonium chloride, coconut trimethyl ammonium bromide, coconut methyldihydroxyethyl ammonium chloride, coconut methyl dihydroxyethyl ammoniumbromide, decyl triethyl ammonium chloride, decyl dimethyl hydroxyethylammonium chloride, decyl dimethyl hydroxyethyl ammonium chloridebromide, C₁₂₋₁₅dimethyl hydroxyethyl ammonium chloride, C₁₂₋₁₅dimethylhydroxyethyl ammonium chloride bromide, coconut dimethyl hydroxyethylammonium chloride, coconut dimethyl hydroxyethyl ammonium bromide,myristyl trimethyl ammonium methyl sulphate, lauryl dimethyl benzylammonium chloride, lauryl dimethyl benzyl ammonium bromide, lauryldimethyl (ethenoxy)₄ ammonium chloride, lauryl dimethyl (ethenoxy)₄ammonium bromide, N-alkyl (C₁₂₋₁₈)dimethylbenzyl ammonium chloride,N-alkyl (C₁₄₋₁₈)dimethyl-benzyl ammonium chloride,N-tetradecylidmethylbenzyl ammonium chloride monohydrate, dimethyldidecyl ammonium chloride, N-alkyl and (C₁₂₋₁₄) dimethyl 1-napthylmethylammonium chloride, trimethylammonium halide, alkyl-trimethylammoniumsalts, dialkyl-dimethylammonium salts, lauryl trimethyl ammoniumchloride, ethoxylated alkyamidoalkyldialkylammonium salt, an ethoxylatedtrialkyl ammonium salt, dialkylbenzene dialkylammonium chloride,N-diclecyldimethyl ammonium chloride, N-tetradecyldimethylbenzylammonium, chloride monohydrate, N-alkyl(C₁₂₋₁₄) dimethyl1-naphthylmethyl ammonium chloride, dodecyldimethylbenzyl ammoniumchloride, dialkyl benzenealkyl ammonium chloride, lauryl trimethylammonium chloride, alkylbenzyl methyl ammonium chloride, alkyl benzyldimethyl ammonium bromide, C₁₂ trimethyl ammonium bromides, C₁₅trimethyl ammonium bromides, C₁₇ trimethyl ammonium bromides,dodecylbenzyl triethyl ammonium chloride, poly-diallyldimethylammoniumchloride (DADMAC), dimethyl ammonium chlorides, alkyldimethylammoniumhalogenides, tricetyl methyl ammonium chloride, decyltrimethylammoniumbromide, dodecyltriethylammonium bromide, tetradecyltrimethylammoniumbromide, methyl trioctylammonium chloride, POLYQUAT 10™,tetrabutylammonium bromide, benzyl trimethylammonium bromide, cholineesters, benzalkonium chloride, stearalkonium chloride compounds, cetylpyridinium bromide, cetyl pyridinium chloride, halide salts ofquaternized polyoxyethylalkylamines, MIRAPOL™, ALKAQUAT™, alkylpyridinium salts; amines, amine salts, amine oxides, imide azoliniumsalts, protonated quaternary acrylamides, methylated quaternarypolymers, cationic guar, polymethylmethacrylate trimethylammoniumbromide, polyvinylpyrrolidone-2-dimetbylaminoethyl methacrylate dimethylsulfate, hexadecyltrimethyl ammonium bromide, poly(2-methacryloxyethyltrimethylammonium bromide) (S1001),poly(N-vinylpyrrolidone/2-dimethylaminoethyl methacrylate) dimethylsulphate quarternary (S1002), (S-630)poly(pyrrolidone-co-vinylacetate) andpoly(2-methylacryloxyamidopropyltrimethylammonium chloride) (S1004).

In some cases the preferred stabilizer is CTAB. Those of skill in theart will appreciate that a wide variety of other surface stabilizers aresuitable for such stabilization.

Should additional purification of the biologically active compound innanoparticulate form be required, then conventional purificationtechniques may be employed. The appropriate technique will depend on thenature of the purification required. Those skilled in the art arefamiliar with such techniques and would readily appreciate adaptation ofsuch techniques to the biologically active compound in nanoparticulateform of the invention.

The present invention includes biologically active compounds innanoparticulate form at least partially separated from the grindingcompound by the methods described above, the use of such in thepreparation of a medicament, and the treatment of an animal, includingman, by the administration of a therapeutically effective amount of thebiologically active compounds in nanoparticulate form by way of themedicament.

A highly advantageous aspect of the present invention is that certaingrinding compounds appropriate for use in the method of the invention(in that they physically degrade to the desired extent under dry millingconditions) are also pharmaceutically acceptable and thus appropriatefor use in a medicament. Where the method of the present invention doesnot involve complete separation of the grinding compound from thebiologically active compound in nanoparticulate form, the presentinvention encompasses methods for the production of a medicamentincorporating both the biologically active compound in nanoparticulateform and at least a portion of the milled grinding compound, medicamentsso produced and methods of treatment of an animal, including man, usinga therapeutically effective amount of said biologically active compoundsby way of said medicaments.

The medicament may include only the biologically active compound innanoparticulate form and the grinding compound or, more preferably, thebiologically active compounds in nanoparticulate form and grindingcompound may be combined with one or more pharmaceutically acceptablecarriers, as well as any desired excipients or other like agentscommonly used in the preparation of medicaments.

Analogously, a highly advantageous aspect of the present invention isthat certain grinding compounds appropriate for use in the method of theinvention (in that they physically degrade to a desirable extent underdry milling conditions) are also appropriate for use in an agriculturalchemical composition. Where the method of the present invention does notinvolve complete separation of the grinding compound from thebiologically active compound in nanoparticulate form, the presentinvention encompasses methods for the production of a agriculturalchemical composition incorporating both the biologically active compoundin nanoparticulate form and at least a portion of the milled grindingcompound, agricultural chemical composition so produced and methods ofuse of such compositions.

The agricultural chemical composition may include only the biologicallyactive compound in nanoparticulate form and the grinding compound or,more preferably, the biologically active compounds in nanoparticulateform and grinding compound may be combined with one or more acceptablecarriers, as well as any desired excipients or other like agentscommonly used in the preparation of agricultural chemical compositions.

In one particular form of the invention, the grinding compound is bothappropriate for use in a medicament and readily separable from thebiologically active compound in nanoparticulate form by methods notdependent on particle size. Such grinding compounds are described in thefollowing detailed description of the invention. Such grinding compoundsare highly advantageous in that they afford significant flexibility inthe extent to which the grinding compound may be incorporated with thebiologically active compound in nanoparticulate form into a medicament.

Thus, the present invention encompasses a method for the manufacture ofa medicament comprising a therapeutically active compound innanoparticulate form, the method comprising the steps of:

-   -   dry milling a mixture of a solid biologically active compound        and a millable grinding compound, in a mill comprising a        plurality of milling bodies, to produce a solid dispersion or        solution comprising nanoparticulate biologically active compound        dispersed in at least partially milled grinding compound; and    -   using said solid dispersion or solution in the manufacture of a        medicament.

The solid dispersion or solution may then be separated from the millingbodies and removed from the mill.

In one embodiment, the grinding compound is separated from thedispersion or solution. Where the grinding compound is not fully milled,the unmilled grinding compound is separated from the nanoparticulatebiologically active compound. In a further aspect, at least a portion ofthe milled grinding compound is separated from the nanoparticulatebiologically active compound.

The milling bodies are essentially resistant to fracture and erosion inthe dry milling process.

The quantity of the grinding compound relative to the quantity ofbiologically active compound in nanoparticulate form, and the extent ofmilling of the grinding compound, is sufficient to inhibitreagglomeration of the biologically active compound in nanoparticulateform.

The grinding compound is not chemically nor mechanically reactive withthe pharmaceutical compound under the dry milling conditions of themethod of the invention.

Preferably, the medicament is a solid dosage form, however, other dosageforms may be prepared by those of ordinary skill in the art.

In one form, after the step of separating said solid solution ordispersion from the plurality of milling bodies, and before the step ofusing said solid solution or dispersion in the manufacture of amedicament, the method may comprise the step of:

-   -   removing a portion of the grinding compound from said solid        dispersion or solution to provide a solid solution or dispersion        enriched in the biologically active compound in nanoparticulate        form;        and the step of using said solid solution or dispersion in the        manufacture of a medicament, more particularly comprises the        step of using the solid solution or dispersion enriched in the        biologically active compound in nanoparticulate form in the        manufacture of a medicament.

In one aspect, where the grinding compound is not fully milled, theunmilled grinding compound is separated from the nanoparticulatebiologically active compound. In a further aspect, at least a portion ofthe milled grinding compound is separated from the nanoparticulatebiologically active compound.

The present invention includes medicaments manufactured by said methods,and methods for the treatment of an animal, including man, by theadministration of a therapeutically effective amount of the biologicallyactive compounds in nanoparticulate form by way of said medicaments.

In another embodiment of the invention, a facilitating agent is alsocomprised in the mixture to be milled. Such facilitating agentsappropriate for use in the invention include diluents, surfacestabilizers, binding agents, filling agents, lubricating agents,sweeteners, flavouring agents, preservatives, buffers, wetting agents,disintegrants, effervescent agents and agents that may form part of amedicament, including a solid dosage form, or other material requiredfor other specific drug delivery, such as the agents and media listedbelow under the heading Medicinal and Pharmaceutical Compositions, orany combination thereof.

A list of examples of surface stabilizers is provided above.

Biologically Active Compounds in Nanoparticulate Form and Compositions

The present invention encompasses pharmaceutically acceptable compoundsin nanoparticulate form produced according to the methods of the presentinvention, compositions including such compounds, including compositionscomprising such compounds together with at least a portion of thegrinding compound.

Where the grinding compound is selectively substantially removed toleave pure pharmaceutically acceptable compounds in nanoparticulateform, agglomeration of the particles may sometimes occur forming largerparticles. Due to the unique nature of the process described, these newagglomerated particles may have unique physical properties, through, forinstance, having new polymorphic structures or nano-structuredmorphologies. Unique polymorphic structures and or the presence of nanostructures morphologies may result in therapeutically beneficialproperties including improved bioavailability. Thus, in some embodimentsof the invention, a composition of the invention comprises substantiallypure pharmaceutically acceptable compounds in nanoparticulate form. Inother embodiments, particularly where the lack of grinding compoundallows the nanoparticles formed during the process to agglomerate in away detrimental to improving the dissolution rate, the preferredcomposition retains at least a portion of the grinding compound.

The pharmaceutically acceptable compounds in nanoparticulate form withinthe compositions of the invention are present at a concentration ofbetween about 0.1% and about 99.0% by weight. Preferably, theconcentration of pharmaceutically acceptable compounds innanoparticulate form within the compositions will be about 5% to about80% by weight, while concentrations of 10% to about 50% by weight arehighly preferred. Desirably, the concentration will be in the range ofabout 10 to 15% by weight, 15 to 20% by weight, 20 to 25% by weight, 25to 30% by weight, 30 to 35% by weight, 35 to 40% by weight, 40 to 45% byweight or 45 to 50% by weight for the composition prior to any laterremoval (if desired) of any portion of the grinding compound. Where partor all of the grinding compound has been removed, the relativeconcentration of pharmaceutically acceptable compounds innanoparticulate form in the composition may be considerably higherdepending on the amount of the grinding compound that is removed. Forexample, if all of the grinding compound is removed the concentration ofnanoparticles in the preparation may be approach 100% by weight (subjectto the presence of facilitating agents).

The dispersion of pharmaceutically acceptable compounds innanoparticulate form in the grinding compound will be dependent on theweight percentage concentration of pharmaceutically acceptable compoundsin nanoparticulate form Depending on that weight percentageconcentration, nanoparticles of the pharmaceutically acceptablecompounds in nanoparticulate form will be “dispersed” in the grindingcompound if at least 0.1% of the nanoparticles are separated by thegrinding compound. Preferably, greater than 10% of the nanoparticleswill be spatially separated from each other by the grinding compound.More preferably at least 15, 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85,90, 92, 95, 98 or 99% of the nanoparticles will be spatially separatedfrom each other by the grinding compound.

Compositions produced according to the present invention are not limitedto the inclusion of a single species of pharmaceutically acceptablecompounds in nanoparticulate form. More than one species ofpharmaceutically acceptable compounds in nanoparticulate form maytherefore be present in the composition. Where more than one species ofpharmaceutically acceptable compounds in nanoparticulate form ispresent, the composition so formed may either be prepared in a drymilling step, or the pharmaceutically acceptable compounds innanoparticulate form may be prepared separately and then combined toform a single composition.

Medicaments

The medicaments of the present invention may include thepharmaceutically acceptable compound in nanoparticulate form, optionallytogether with at least a portion of the grinding compound, combined withone or more pharmaceutically acceptable carriers, as well as otheragents commonly used in the preparation of pharmaceutically acceptablecompositions.

As used herein “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forparenteral administration, intravenous, intraperitoneal, intramuscular,sublingual, transdermal or oral administration. Pharmaceuticallyacceptable carriers include sterile aqueous solutions or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. The use of such media and agents for themanufacture of medicaments is well known in the art. Except insofar asany conventional media or agent is incompatible with thepharmaceutically acceptable compound in nanoparticulate form, usethereof in the manufacture of a pharmaceutical composition according tothe invention is contemplated.

Pharmaceutical acceptable carriers according to the invention mayinclude one or more of the following examples:

-   -   (1) polymeric surface stabilizers which are capable of adhering        to the surface of the active agent but do not take part in or        undergo any chemical reaction with the active agent itself, such        as polymeric surface stabilizers, including, but are not limited        to polyethylene glycol (PEG), polyvinylpyrrolidone (PVP),        polyvinylalcohol, corspovidone,        polyvinylpyrrolidone-polyvinylacytate copolymer, cellulose        derivatives, hydroxypropylmethyl cellulose, hydroxypropyl        cellulose, carboxymethylethyl cellulose, hydroxypropyllmethyl        cellulose phthalate, polyacrylates and polymethacrylates, urea,        sugars, polyols, and their polymers, emulsifiers, sugar gum,        starch, organic acids and their salts, vinyl pyrrolidone and        vinyl acetate; and or    -   (2) binding agents such as various celluloses and cross-linked        polyvinylpyrrolidone, microcrystalline cellulose; and or    -   (3) filling agents such as lactose monohydrate, lactose        anhydrous, and various starches; and or    -   (4) lubricating agents such as agents that act on the        flowability of the powder to be compressed, including colloidal        silicon dioxide, talc, stearic acid, magnesium stearate, calcium        stearate, silica gel; and or    -   (5) sweeteners such as any natural or artificial sweetener        including sucrose, xylitol, sodium saccharin, cyclamate,        aspartame, and accsulfame K; and or    -   (6) flavouring agents; and or    -   (7) preservatives such as potassium sorbate, methylparaben,        propylparaben, benzoic acid and its salts, other esters of        parahydroxybenzoic acid such as butylparaben, alcohols such as        ethyl or benzyl alcohol, phenolic compounds such as phenol, or        quarternary compounds such as benzalkonium chloride; and or    -   (8) buffers; and or    -   (9) Diluents such as pharmaceutically acceptable inert fillers,        such as microcrystalline cellulose, lactose, dibasic calcium        phosphate, saccharides, and/or mixtures of any of the foregoing;        and or    -   (10) wetting agents such as corn starch, potato starch, maize        starch, and modified starches, croscarmellose sodium,        crosspovidone, sodium starch glycolate, and mixtures thereof;        and or    -   (11) disintegrants; and or    -   (12) effervescent agents such as effervescent couples such as an        organic acid (e.g., citric, tartaric, malic, fumaric, adipic,        succinic, and alginic acids and anhydrides and acid salts), or a        carbonate (e.g. sodium carbonate, potassium carbonate, magnesium        carbonate, sodium glycine carbonate, L-lysine carbonate, and        arginine carbonate) or bicarbonate (e.g. sodium bicarbonate or        potassium bicarbonate); and or    -   (13) other pharmaceutically acceptable excipients.

Medicaments of the invention suitable for use in animals and inparticular in man typically must be sterile and stable under theconditions of manufacture and storage. The medicaments of the inventioncomprising the biologically active compound in nanoparticulate form canbe formulated as a solution, microemulsion, liposome, or other orderedstructure suitable to high drug concentration. Actual dosage levels ofthe biologically active compound in the medicament of the invention maybe varied in accordance with the nature of the biologically activecompound, as well as the potential increased efficacy due to theadvantages of providing and administering the biologically activecompound in nanoparticulate form (e.g., increased solubility, more rapiddissolution, increased surface area of the biologically active compoundin nanoparticulate form, etc.). Thus as used herein “therapeuticallyeffective amount” will refer to an amount of biologically activecompound in nanoparticulate form required to effect a therapeuticresponse in an animal. Amounts effective for such a use will depend on:the desired therapeutic effect; the route of administration; the potencyof the biologically active compound; the desired duration of treatment;the stage and severity of the disease being treated; the weight andgeneral state of health of the patient; and the judgment of theprescribing physician.

In another embodiment, the biologically active compound innanoparticulate form, optionally together with at least a portion of thegrinding compound, of the invention may be combined into a medicamentwith another biologically active compound, or even the same biologicallyactive compound. In the latter embodiment, a medicament may be achievedwhich provides for different release characteristics—early release fromthe biologically active compound in nanoparticulate form, and laterrelease from a larger average size biologically active compound innanoparticulate form or a non-nanoparticulate biologically activecompound.

Modes of Administration of Medicaments Comprising Biologically ActiveCompounds in Nanoparticulate Form

Medicaments of the invention can be administered to animals, includingman, in any pharmaceutically acceptable manner, such as orally,rectally, pulmonary, intravaginally, locally (powders, ointments ordrops), transdermal, or as a buccal or nasal spray.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, pellets, and granules. For capsules, tablets, and pills,the dosage forms may also comprise buffering agents. Further,incorporating any of the normally employed excipients, such as thosepreviously listed, and generally 10-95% of the biologically active agentin nanoparticulate form, and more preferably at a concentration of25%-75% will form a pharmaceutically acceptable non-toxic oralcomposition.

Medicaments of the invention may be parenterally administered as asolution of the biologically active agent in nanoparticulate formsuspended in an acceptable carrier, preferably an aqueous carrier. Avariety of aqueous carriers may be used, e.g. water, buffered water,0.4% saline, 0.3% glycine, hyaluronic acid and the like. Thesecompositions may be sterilized by conventional, well known sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile solution prior toadministration.

For aerosol administration, medicaments of the invention are preferablysupplied along with a surface stabilizer and propellant. The surfacestabilizer must, of course, be non-toxic, and preferably soluble in thepropellant. Representative of such agents are the esters or partialesters of fatty acids containing from 6 to 22 carbon atoms, such ascaproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic,olesteric and oleic acids with an aliphatic polyhydric alcohol or itscyclic anhydride. Mixed esters, such as mixed or natural glycerides maybe employed. The surface stabilizer may constitute 0.1%-20% by weight ofthe composition, preferably 0.25-5%. The balance of the composition isordinarily propellant. A carrier can also be included, as desired, aswith, e.g., lecithin for intranasal delivery.

Medicaments of the invention may also be administered via liposomes,which serve to target the active agent to a particular tissue, such aslymphoid tissue, or targeted selectively to cells. Liposomes includeemulsions, foams, micelles, insoluble monolayers, liquid crystals,phospholipid dispersions, lamellar layers and the like. In thesepreparations the nanocomposite microstructure composition isincorporated as part of a liposome, alone or in conjunction with amolecule that binds to or with other therapeutic or immunogeniccompositions.

As described above, the biologically active compound in nanoparticulateform can be formulated into a solid dosage form (e.g., for oral orsuppository administration), together with at least a portion of thegrinding compound then there may be little or no need for furtherstabilizing the dispersion since the grinding compound may effectivelyact as a solid-state stabilizer.

However, if the biologically active compound in nanoparticulate form isto be utilized in a liquid (or gaseous) suspension, the nanoparticlescomprising the biologically active compound in nanoparticulate form mayrequire further stabilization once the solid carrier has beensubstantially removed to ensure the elimination, or at leastminimisation of particle agglomeration.

Therapeutic Uses

Therapeutic uses of the medicaments of the invention include painrelief, anti-inflammatory, migraine, asthma, and other disorders thatrequire the active agent to be administered with a high bioavailability.

One of the main areas when rapid bioavailability of a biologicallyactive compound is required is in the relief of pain. The minoranalgesics, such as cyclooxgenase inhibitors (aspirin related drugs) maybe prepared as medicaments according to the present invention.

Medicaments of the invention may also be used for treatment of eyedisorders. That is, the biologically active compound in nanoparticulateform may be formulated for administration on the eye as an aqueoussuspension in physiological saline, or a gel. In addition, thebiologically active compound in nanoparticulate form may be prepared ina powder form for administration via the nose for rapid central nervoussystem penetration.

Treatment of cardiovascular disease may also benefit from biologicallyactive compounds in nanoparticulate form according to the invention,such as treatment of angina pectoris and, in particular, molsidomine maybenefit from better bioavailability.

Other therapeutic uses of the medicaments of the present inventioninclude treatment of hair loss, sexual dysfunction, or dermal treatmentof psoriasis.

The invention will now be described with greater particularity for thepreparation of forms of raloxifene.

Forms of Raloxifene

The present invention encompasses particulate amorphous raloxifene,pharmaceutically acceptable raloxifene salts and solvates. Methods forthe preparation of such amorphous compounds are described in U.S. Pat.No. 6,713,494 (Eli Lilly and Company).

Where the raloxifene or the pharmaceutically acceptable salt or solvateof the present invention is crystalline, the present invention shouldnot be understood to be limited to any particular polymorph thereof.

Pharmaceutically acceptable salts of the present invention may be formedfrom a range of organic or inorganic acids.

Typical inorganic acids used to form such salts include hydrochloric,hydrobromic, hydroiodic, nitric, sulfuric, phosphoric, hypophosphoric,and the like.

Salts derived from organic acids, such as aliphatic mono anddicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoicand hydroxyalkandioic acids, aromatic acids, aliphatic and aromaticsulfonic acids, may also be used. Such pharmaceutically acceptable saltsthus include acetate, phenylacetate, trifluoroacetate, acrylate,ascorbate, benzoate, chlorobenzoate, di nitrobenzoate, hydroxybenzoate,methoxybenzoate, methylbenzoate, o-acetoxybenzoate,naphthalene-2-benzoate, bromide, isobutyrate, phenylbutyrate,hydroxybutyrate, butyne-1,4-dioate, hexyne-1,4-dioate, caproate,caprylate, chloride, cinnamate, citrate, formate, fumarate, glycolate,heptanoate, hippurate, lactate, malate, maleate, hydroxymaleate,malonate, mandelate, mesylate, nicotinate, isonicotinate, nitrate,oxalate, phthalate, terephthalate, phosphate, monohydrogenphosphate,dihydrogenphosphate, metaphosphate, pyrophosphate, propiolate,propionate, phenylpropionate, salicylate, sebacate, succinate, suberate,sulfate, bisulfate, pyrosulfate, sulfite, bisulfite, sulfonate,benzenesulfonate, p-bromophenylsulfonate, chlorobenzenesulfonate,ethanesulfonate, 2-hydroxyethanesulfonate, methanesulfonate,naphthalene-1-sulfonate, naphthalene-2-sulfonate, p-toluenesulfonate,xylenesulfonate, tartrate, and the like.

Published US patent application 20060154966 describes the preparation ofraloxifene D-lactate, raloxifene L-lactate, Raloxifene DL-lactate,raloxifene D-lactate hemihydrate, raloxifene D-lactate ¼-hydrate,raloxifene L-lactate hemihydrate, raloxifene L-lactate ¼-hydrate,raloxifene DL-lactate hemihydrate and raloxifene DL-lactate ¼-hydrate.

For certain applications, a preferred salt is the hydrochloride salt.

Particle Size

The costs associated with reducing particle size are not limited to thedirect cost of milling. For example, U.S. Pat. No. 6,894,064 (Eli Lillyand Company; Benzothiapenes, formulations containing same and methods)explains that “very finely divided material presents difficulties andcosts in capsule filling or tablet preparation, because the materialwill not flow, but becomes caked in finishing machinery”, and that“[s]uch finishing difficulties generate non-homogeneity in the finalproduct, which is not acceptable in a drug substance”. Accordingly“there is always a dynamic between the properties which yield themaximum bioavailability (particle surface area) and the practical limitsof manufacture” and “[t]he point of compromise which marks the “bestsolution” is unique to each situation and unique as to itsdetermination”.

It has now been found that by processing raloxifene, or apharmaceutically acceptable salt or solvate thereof, to bring theparticle size within the specified range, pharmaceutical compositionsmay be prepared which exhibit improved in vitro dissolution profiles andin vivo bioavailability relative to some known raloxifene hydrochlorideforms. Further, in some forms of the invention, these improvements maybe achieved without importing characteristics that are disadvantageousfrom a manufacturing perspective.

As stated above, the invention is characterised in that the particulateraloxifene, or pharmaceutically acceptable salt or solvate thereof, inparticulate form having a mean particle size of between about 10 nm andabout 500 nm.

In one form of the invention, the mean particle size is between about 75nm and about 500 nm. In one form of the invention, the mean particlesize is between about 75 nm and about 400 nm. In one form of theinvention, the mean particle size is between about 75 nm and about 300nm. In one form of the invention, the mean particle size is betweenabout 75 nm and about 200 nm. In one form of the invention, the meanparticle size is between about 75 nm and about 100 nm.

Size Distribution

In preferred forms of the invention, the particulate raloxifene,pharmaceutically acceptable salt or solvate thereof, has a narrowparticle size distribution.

In a preferred form of the invention, about 90% of the particles have aparticle size of less than about 500 nm. The particles distribution canbe measured with dynamic light scattering of a dispersion of theparticles with the aid of sonication, and after centrifugation at 500rcf for 30 seconds to remove larger agglomerates from the dispersion.Other means to measure the particles size are for examples surface areameasurements, and electron micrographs, which can be used to support themeasured size distribution of the particles.

In one form of the invention, about 50% of the particles have a particlesize of less than 500 nm. In another form of the invention, about 90% ofthe particles have a particle size of less than 500 nm. In one form ofthe invention, about 90% of the particles have a particle size ofbetween about 100 and 500 nm. In one form of the invention, about 90% ofthe particles have a particle size of between about 75 nm and about 500nm. In one form of the invention, about 90% of the particles have aparticle size of between about 75 nm and about 400 nm. In one form ofthe invention, about 90% of the particles have a particle size ofbetween about 75 nm and about 300 nm. In one form of the invention,about 90% of the particles have a particle size of between about 75 nmand about 200 nm. In one form of the invention, about 90% of theparticles have a particle size of between about 75 nm and about 100 nm.

In addition to the role of particle size in vitro dissolution and invivo absorption, another important aspect is its role on the variousoperations of the drug product manufacturing process. While the particlesize specification ensures consistent delivery of the drug molecule tothe sites of absorption in the gastrointestinal tract, it also allowsfor better control during the wet granulation step of the tabletmanufacturing process.

By controlling the particle size, the variations in quantity of waterneeded to elicit the appropriate progression of the granulation powerconsumption curve is reduced. By maintaining the particle size withinthe previous mentioned constraints, established quantities of water canbe dictated in the manufacturing ticket for routine lot manufacture. Thegranulation step is common to many tablet and capsule manufacturingoperations and is typically driven by the addition of water to bringabout the desired endpoint of the granulation. A downstream unitoperation dependent upon the granulation endpoint is the milling of thedried granulation and the resulting particle size distribution obtainedon the granulation. It has been discovered that the incoming particlesize of the active ingredient also effects the ultimate particle sizedistribution of the dry milled agglomerates formed during granulations.For a fixed water quantity, a coarser distribution will result in afiner size distribution of the dry milled agglomerates. Too fine agranulation distribution can lead to poor granulation flow and poorcontrol of individual tablet weight during the compression step. Thusthe narrow particle size constraints previously mentioned have alsoresulted in making the process more amenable to automation by reducingthe variations in water required during the granulation step andproducing dry milled granules of the appropriate distribution to preventthe rejection of tablets during compression due to unacceptable tabletweight.

Agglomerates

Agglomerates comprising particles of raloxifene, a pharmaceuticallyacceptable salt or solvate thereof, said particles having a meanparticle size of between about 10 nm and about 500 nm, should beunderstood to fall within the scope of the present invention, regardlessof whether the agglomerates exceed 500 nm in size.

For certain applications of the particulate raloxifene, pharmaceuticallyacceptable salt or solvate thereof, of the present invention, theformation of agglomerates is highly desirable. Agglomerates of particlesof raloxifene, or a pharmaceutically acceptable salt or solvate thereof,of the invention may afford the advantages of improved in vitrodissolution and in vivo bioavailability relative to some knownraloxifene hydrochloride forms without attracting the processingdisadvantages conventionally associated with decreased particle sizes.

Other Properties

In preferred forms of the invention, the particulate raloxifene,pharmaceutically acceptable salt or solvate thereof has a surface areaof in excess of 5 m²/g. Preferably still the particulate raloxifene,pharmaceutically acceptable salt or solvate thereof has a surface areain excess of 7 m²/g. Preferably still the particulate raloxifene,pharmaceutically acceptable salt or solvate thereof has a surface areain excess of 10 m²/g. Preferably still the particulate raloxifene,pharmaceutically acceptable salt or solvate thereof has a surface areain excess of 15 m²/g. Preferably still the particulate raloxifene,pharmaceutically acceptable salt or solvate thereof has a surface areain excess of 20 m²/g. Preferably still the particulate raloxifene,pharmaceutically acceptable salt or solvate thereof has a surface areain excess of 25 m²/g. Preferably still the particulate raloxifene,pharmaceutically acceptable salt or solvate thereof has a surface areain excess of 30 m²/g. Preferably still the particulate raloxifene,pharmaceutically acceptable salt or solvate thereof has a surface areain excess of 35 m²/g. Preferably still the particulate raloxifene,pharmaceutically acceptable salt or solvate thereof has a surface areain excess of 40 m²/g. Preferably still the particulate raloxifene,pharmaceutically acceptable salt or solvate thereof has a surface areain excess of 50 m²/g. Preferably still the particulate raloxifene,pharmaceutically acceptable salt or solvate thereof has a surface areain excess of 55 m²/g. Preferably still the particulate raloxifene,pharmaceutically acceptable salt or solvate thereof has a surface areaof up to approximately 57 m²/g.

In one aspect of the invention, there is provided particulatecrystalline raloxifene, or a pharmaceutically acceptable salt or solvatethereof which, when administered orally to dogs, demonstrates a peakplasma concentration (C_(max)) of greater than 12 ng/m L.

In one aspect of the invention, there is provided particulatecrystalline raloxifene, or a pharmaceutically acceptable salt or solvatethereof which, when administered orally to dogs, demonstrates an areaunder the concentration versus time curve (AUC_(0-t)) greater than 33ng·h/mL.

In one aspect of the invention, there is provided particulatecrystalline raloxifene, or a pharmaceutically acceptable salt or solvatethereof which, when administered orally to dogs, demonstrates a mediantime to maximum plasma concentration (T_(max)) of within 1 hour.

Methods of Producing Particulate Raloxifene Hydrochloride

As summarised, the present invention further provides methods forproducing said particulate raloxifene, or a pharmaceutically acceptablesalt or solvate thereof.

In particular, the present invention comprises a method for producing aparticulate raloxifene, or a pharmaceutically acceptable salt or solvatethereof, with a mean particle size of between about 10 nm and about 500nm, the method comprising the step of:

-   -   milling a mixture of a solid raloxifene hydrochloride and a        millable grinding compound, in a mill comprising a plurality of        milling bodies, to produce a solid dispersion or solution        comprising particulate raloxifene or a pharmaceutically        acceptable salt or solvate thereof with a mean particle size of        between about 10 nm and about 500 nm dispersed in at least        partially milled grinding compound.

In a preferred form of the invention, the milling step is a dry millingstep.

In one form of the invention, the mean particle size is between about 75nm and about 500 nm. In one form of the invention, the mean particlesize is between about 75 nm and about 400 nm. In one form of theinvention, the mean particle size is between about 75 nm and about 300nm. In one form of the invention, the mean particle size is betweenabout 75 nm and about 200 nm. In one form of the invention, the meanparticle size is between about 75 nm and about 100 nm.

In a preferred form of the invention, about 50% of the particles have aparticle size of less than about 500 nm. In one form of the invention,about 90% of the particles have a particle size of between about 100 and500 nm. In one form of the invention, about 90% of the particles have aparticle size of between about 75 nm and about 500 nm. In one form ofthe invention, about 90% of the particles have a particle size ofbetween about 75 nm and about 400 nm. In one form of the invention,about 90% of the particles have a particle size of between about 75 nmand about 300 nm. In one form of the invention, about 90% of theparticles have a particle size of between about 75 nm and about 200 nm.In one form of the invention, about 90% of the particles have a particlesize of between about 75 nm and about 100 nm.

The term “millable” means that the grinding compound is capable of beingphysically degraded under the dry milling conditions of the method ofthe invention. In one embodiment of the invention, the milled grindingcompound is of a comparable particle size to the nanoparticulatebiologically active compound.

In a highly preferred form, the grinding compound is harder than thesolid raloxifene, pharmaceutically acceptable salt or solvate thereof,and is thus capable of physically degrading such under the dry millingconditions of the invention. Again without wishing to be bound bytheory, under these circumstances it is believed that the millablegrinding compound affords the advantage of the present invention througha second route, with the smaller particles of grinding compound producedunder the dry milling conditions enabling the production of smallerparticles of raloxifene or the pharmaceutically acceptable salt orsolvate thereof.

The solid dispersion or solution of raloxifene or the pharmaceuticallyacceptable salt or solvate thereof may then be separated from themilling bodies and removed from the mill.

In a preferred aspect, the grinding compound is separated from thedispersion or solution. In a further aspect, at least a portion of themilled grinding compound is separated from the particulate raloxifenehydrochloride.

The milling bodies are essentially resistant to fracture and erosion inthe dry milling process.

The quantity of the grinding compound relative to the quantity ofparticulate raloxifene, pharmaceutically acceptable salt or solvatethereof, and the extent of physical degradation of the grindingcompound, is sufficient to inhibit reagglomeration of the raloxifene,pharmaceutically acceptable salt or solvate thereof. The grindingcompound is not chemically reactive with the pharmaceutical raloxifene,pharmaceutically acceptable salt or solvate thereof under the millingconditions of the invention.

In one embodiment of the invention, the milled grinding compound is of acomparable particle size to the particulate raloxifene, pharmaceuticallyacceptable salt or solvate thereof.

The physical properties of the grinding compound necessary to achievethe requisite degradation are dependant on the precise millingconditions. For example a harder grinding compound may degrade to asufficient extent provided under more vigorous dry milling conditions.

Physical properties of the grinding compound relevant to the extent thatthe agent will degrade under dry milling conditions include hardness,friability, as measured by indicia such as fracture toughness andbrittleness index.

Preferably, the grinding compound is of low abrasivity. Low abrasivityis desirable to minimise contamination of the dispersion or solution ofthe particulate raloxifene hydrochloride in the grinding compound by themilling bodies and/or the milling chamber of the media mill. An indirectindication of the abrasivity can be obtained by measuring the level ofmilling-based contaminants.

Preferably, the grinding compound has a low tendency to agglomerateduring dry milling. While it is difficult to objectively quantify thetendency to agglomerate during milling, it is possible to obtain asubjective measure by observing the level of “caking” of the grindingcompound on the milling bodies and the milling chamber of the media millas dry milling progresses.

The grinding compound may be an inorganic or organic compound. In oneembodiment, the grinding compound is selected from the following: sodiumhydrogen sulfate, sodium hydrogen carbonate, sodium hydroxide, orsuccinic acid; crystalline organic acids, for example (but not limitedto) fumaric acid, maleic acid, tartaric acid, citric acid);alternatively ammonium salts (or salts of volatile amines), for example(but not limited to) ammonium chloride, methylamine hydrochloride,ammonium bromide, crystalline hydroxides, hydrogen carbonates, hydrogencarbonates of pharmaceutical acceptable alkali metals, such as but notlimited by, sodium, potassium, lithium, calcium, and barium, sodiumsulphate, sodium chloride, sodium metabisulphite, sodium thiosulphate,ammonium chloride, Glauber's salt, ammonium carbonate, sodiumbisulphate, magnesium sulphate, potash alum, potassium chloride, sodiumcarbonate, sodium bicarbonate, potassium carbonate, potassiumbicarbonate.

Selection of an appropriate grinding compound affords particular highlyadvantageous applications of the method of the present invention. Somegrinding compounds appropriate for use in the invention are readilyseparable from the particulate raloxifene, pharmaceutically acceptablesalt or solvate thereof by methods not dependent on particle size (suchmethods being inappropriate due to the degradation of the grindingcompound). For example, selecting an appropriate grinding compound thatalso possesses solubility properties different from the particulateraloxifene, pharmaceutically acceptable salt or solvate thereof allowsseparation of the two by relatively straightforward selectivedissolution techniques. Examples of such grinding compounds are providedin the detailed description of the invention. Thus, a particularlyadvantageous application of the method of the invention is the use of awater-soluble salt as a grinding compound.

A highly advantageous aspect of the present invention is that certaingrinding compounds appropriate for use in the method of the inventionare also appropriate for use in a medicament. The present inventionencompasses methods for the production of a medicament incorporatingboth the particulate raloxifene, pharmaceutically acceptable salt orsolvate thereof and at least a portion of the grinding compound,medicaments so produced, and methods of treatment of an animal,including man, using a therapeutically effective amount of saidbiologically active compounds by way of said medicaments.

The medicament may include only the particulate raloxifene,pharmaceutically acceptable salt or solvate thereof together with themilled grinding compound or, more preferably, the particulateraloxifene, pharmaceutically acceptable salt or solvate thereof andmilled grinding compound may be combined with one or morepharmaceutically acceptable carriers, as well as any desired excipientsor other like agents commonly used in the preparation of medicaments.

In one particular form of the invention, the grinding compound is bothappropriate for use in a medicament and readily separable from theparticulate raloxifene, pharmaceutically acceptable salt or solvatethereof by methods not dependent on particle size. Such grindingcompounds are described in the following detailed description of theinvention. Such grinding compounds are highly advantageous in that theyafford significant flexibility in the extent to which the grindingcompound may be incorporated with the particulate raloxifene,pharmaceutically acceptable salt or solvate thereof into a medicament.

In one form of the invention, the grinding compound is sodium chloride.In one form of the invention, the grinding compound is calciumcarbonate.

In a preferred embodiment, the grinding compound is a compound that isconsidered GRAS (generally regarded as safe) by persons skilled in thepharmaceutical arts.

In a preferred form of the invention, prior to the step of:

-   -   dry milling a mixture of a solid raloxifene hydrochloride and a        millable grinding compound, in a mill comprising a plurality of        milling bodies, to produce a solid dispersion or solution        comprising particulate raloxifene or a pharmaceutically        acceptable salt or solvate thereof with a mean particle size of        between about 10 nm and about 500 nm dispersed in at least        partially milled grinding compound;        the method of the present invention comprises the step of        substantially drying the solid raloxifene or pharmaceutically        acceptable salt or solvate thereof.

Preferably still, prior to the step of:

-   -   dry milling a mixture of a solid raloxifene hydrochloride and a        millable grinding compound, in a mill comprising a plurality of        milling bodies, to produce a solid dispersion or solution        comprising particulate raloxifene or a pharmaceutically        acceptable salt or solvate thereof with a mean particle size of        between about 10 nm and about 500 nm dispersed in at least        partially milled grinding compound;        the method of the present invention comprises the step of        substantially drying the grinding compound.

Persons skilled in the art will be aware of many techniques for removingwater from compounds. In one form of the invention, the step ofsubstantially drying the solid raloxifene, pharmaceutically acceptablesalt or solvate, is performed by exposing the raloxifene hydrochlorideto a drying agent under vacuum for a suitable period of time. Personsskilled in the art will be aware of a range of appropriate dryingagents.

In one form of the invention the drying agent is P₂O₅.

The number and size of milling bodies can be varied to alter the amountof energy applied during the milling. This results in variation of thesize and characteristics of the resultant raloxifene. The examples showcertain combinations optimized for the current scale of manufacture,however those skilled in the art will appreciate that as the process isscaled, variations to milling media size, number and energy applied willbe required to produce the same product.

Preferably, the concentration of solid raloxifene, pharmaceuticallyacceptable salt or solvate, in the mixture of solid raloxifene solidraloxifene, pharmaceutically acceptable salt or solvate, and themillable grinding compound is between about 5% and about 25% v/v.Preferably still, the concentration is between about 5% and about 20%v/v. In a highly preferred form of the invention, the concentration isbetween about 10% and about 15% v/v. In one form of the invention, theconcentration is about 15% v/v.

Milling Bodies

In the method of the present invention, the milling bodies arepreferably chemically inert and rigid. The term “chemically-inert”, asused herein, means that the milling bodies do not react chemically withthe rolixifene hydrochloride or the grinding compound.

The milling bodies are essentially resistant to fracture and erosion inthe milling process.

The milling bodies are desirably provided in the form of bodies whichmay have any of a variety of smooth, regular shapes, flat or curvedsurfaces, and lacking sharp or raised edges. For example, suitablemilling bodies can be in the form of bodies having ellipsoidal, ovoid,spherical or right cylindrical shapes. Preferably, the milling bodiesare provided in the form of one or more of beads, balls, spheres, rods,right cylinders, drums or radius-end right cylinders (i.e., rightcylinders having hemispherical bases with the same radius as thecylinder).

The milling media bodies desirably have an effective mean particlediameter (i.e. “particle size”) between about 0.1 and 30 mm, morepreferably between about 1 and about 15 mm, still more preferablybetween about 3 and 10 mm.

The milling bodies may comprise various materials such as ceramic,glass, metal or polymeric compositions, in a particulate form. Suitablemetal milling bodies are typically spherical and generally have goodhardness (i.e. RHC 60-70), roundness, high wear resistance, and narrowsize distribution and can include, for example, balls fabricated fromtype 52100 chrome steel, type 316 or 440C stainless steel or type 1065high carbon steel.

Preferred ceramic materials, for example, may be selected from a widearray of ceramics desirably having sufficient hardness and resistance tofracture to enable them to avoid being chipped or crushed during millingand also having sufficiently high density. Suitable densities formilling media can range from about 1 to 15 g/cm³. Preferred ceramicmaterials can be selected from steatite, aluminum oxide, zirconiumoxide, zirconia-silica, yttria-stabilized zirconium oxide,magnesia-stabilized zirconium oxide, silicon nitride, silicon carbide,cobalt-stabilized tungsten carbide, and the like, as well as mixturesthereof.

Preferred glass milling media are spherical (e.g. beads), have a narrowsize distribution, are durable, and include, for example, lead-free sodalime glass and borosilicate glass. Polymeric milling media arepreferably substantially spherical and can be selected from a wide arrayof polymeric resins having sufficient hardness and friability to enablethem to avoid being chipped or crushed during milling,abrasion-resistance to minimize attrition resulting in contamination ofthe product, and freedom from impurities such as metals, solvents, andresidual monomers.

Preferred polymeric resins, for example, can be selected fromcrosslinked polystyrenes, such as polystyrene crosslinked withdivinylbenzene, styrene copolymers, polyacrylates such aspolymethylmethacrylate, polycarbonates, polyacetals, vinyl chloridepolymers and copolymers, polyurethanes, polyamides, high densitypolyethylenes, polypropylenes, and the like. The use of polymericmilling media to grind materials down to a very small particle size (asopposed to mechanochemical synthesis) is disclosed, for example, in U.S.Pat. Nos. 5,478,705 and 5,500,331. Polymeric resins typically can havedensities ranging from about 0.8 to 3.0 g/cm³. Higher density polymericresins are preferred. Alternatively, the milling media can be compositeparticles comprising dense core particles having a polymeric resinadhered thereon. Core particles can be selected from materials known tobe useful as milling media, for example, glass, alumina, zirconiasilica, zirconium oxide, stainless steel, and the like. Preferred corematerials have densities greater than about 2.5 g/cm³.

In one embodiment of the invention, the milling media are formed from aferromagnetic material, thereby facilitating removal of contaminantsarising from wear of the milling media by the use of magnetic separationtechniques.

Each type of milling body has its own advantages. For example, metalshave the highest specific gravities, which increase grinding efficiencydue to increased impact energy. Metal costs range from low to high, butmetal contamination of final product can be an issue. Glasses areadvantageous from the standpoint of low cost and the availability ofsmall bead sizes as low as 0.004 mm. However, the specific gravity ofglasses is lower than other media and significantly more milling time isrequired. Finally, ceramics are advantageous from the standpoint of lowwear and contamination, ease of cleaning, and high hardness.

In a specific form of the invention, the milling bodies comprise aplurality of steel balls of approximately 3 cm³ volume and 40 g mass.

Dry Milling

In the dry milling process of the present invention, the solidraloxifene, or pharmaceutically acceptable salt or solvate thereof, andgrinding compound, in the form of crystals, powders, or the like, arecombined in suitable proportions with the plurality of milling bodies ina milling chamber that is mechanically agitated (i.e., with or withoutstirring) for a predetermined period of time at a predeterminedintensity of agitation. Typically, a milling apparatus is used to impartmotion to the milling bodies by the external application of agitation,whereby various translational, rotational or inversion motions orcombinations thereof are applied to the milling chamber and itscontents, or by the internal application of agitation through a rotatingshaft terminating in a blade, propeller, impeller or paddle or by acombination of both actions.

During milling, motion imparted to the milling bodies can result inapplication of shearing forces as well as multiple impacts or collisionshaving significant intensity between milling bodies and particles of thereactant powders. The solid raloxifene hydrochloride and the grindingcompound is influenced by a wide variety of processing parametersincluding: the type of milling apparatus; the intensity of the forcesgenerated, the kinematic aspects of the process; the size, density,shape, and composition of the milling bodies; the weight ratio of theraloxifene hydrochloride and grinding compound mixture to the millingbodies; the duration of milling; the physical properties the grindingcompound; the atmosphere present during activation; and others.

Advantageously, the media mill is capable of repeatedly or continuouslyapplying mechanical compressive forces and shear stress to thebiologically active compound substrate and the grinding compound.Suitable media mills include but are not limited to the following:high-energy ball, sand, bead or pearl mills, basket mill, planetarymill, vibratory action ball mill, multi-axial shaker/mixer, stirred ballmill, horizontal small media mill, multi-ring pulverizing mill, and thelike, including small milling media. The milling apparatus also cancontain one or more rotating shafts.

In a preferred form of the invention, the dry milling is effected in aball mill. Throughout the remainder of the specification reference willbe made to dry milling being carried out by way of a ball mill. Examplesof this type of mill are attritor mills, nutating mills, tower mills,planetary mills, vibratory mills and gravity-dependent-type ball mills.It will be appreciated that dry milling in accordance with the method ofthe invention may also be achieved by any suitable means other than ballmilling. For example, dry milling may also be achieved using jet mills,rod mills, roller mills or crusher mills.

It is preferred, but not essential, that the particle size of the solidraloxifene, or pharmaceutically acceptable salt or solvate thereof beless than about 100 μm, as may be determined by sieve analysis. If thecoarse particle size of the solid raloxifene or pharmaceuticallyacceptable salt or solvate thereof, is greater than about 100 μm, thenit is preferred that the particles of the solid raloxifene, orpharmaceutically acceptable salt or solvate thereof, be first reduced insize to less than 100 μm using a conventional milling method such asairjet or fragmentation milling.

Pharmaceutical Compositions Comprising, or Formulated Using, ParticulateRaloxifene, or Pharmaceutically Acceptable Salt or Solvate Thereof,According to the Invention

As stated in the summary, the present invention also providespharmaceutical compositions comprising or formulated using the saidparticulate raloxifene, or pharmaceutically acceptable salt or solvatethereof.

The pharmaceutical compositions of the invention may include apharmaceutically acceptable carrier, wherein “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like that are physiologically compatible.

Preferably, the pharmaceutically acceptable carrier is suitable forparenteral, intravenous, intraperitoneal, intramuscular, sublingual,transdermal or oral administration. Pharmaceutically acceptable carriersinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersion. The use of such media and agents for the manufacture ofpharmaceutical compositions is well known in the art. Except insofar asany conventional media or agent is incompatible with the particulateraloxifene hydrochloride of the invention, use thereof in themanufacture of a pharmaceutical composition according to the inventionis contemplated.

Pharmaceutical compositions according to the invention may include oneor more of the following additives:

-   (1) polymeric surface stabilizers which are capable of adhering to    the surface of the active agent but do not take part in or undergo    any chemical reaction with the active agent itself, such as    polymeric surface stabilizers, including, but are not limited to    polyethylene glycol (PEG), polyvinylpyrrolidone (PVP),    polyvinylalcohol, corspovidone,    polyvinylpyrrolidone-polyvinylacytate copolymer, cellulose    derivatives, hydroxypropylmethyl cellulose, hydroxypropyl cellulose,    carboxymethylethyl cellulose, hydroxypropyllmethyl cellulose    phthalate, polyacrylates and polymethacrylates, urea, sugars,    polyols, and their polymers, emulsifiers, sugar gum, starch, organic    acids and their salts, vinyl pyrrolidone and vinyl acetate; and or-   (2) binding agents such as various celluloses and cross-linked    polyvinylpyrrolidone, microcrystalline cellulose; and or-   (3) filling agents such as lactose monohydrate, lactose anhydrous,    and various starches; and or-   (4) lubricating agents such as agents that act on the flowability of    the powder to be compressed, including colloidal silicon dioxide,    talc, stearic acid, magnesium stearate, calcium stearate, silica    gel; and or-   (5) sweeteners such as any natural or artificial sweetener including    sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and    accsulfame K; and or-   (6) flavouring agents; and or-   (7) preservatives such as potassium sorbate, methylparaben,    propylparaben, benzoic acid and its salts, other esters of    parahydroxybenzoic acid such as butylparaben, alcohols such as ethyl    or benzyl alcohol, phenolic compounds such as phenol, or quarternary    compounds such as benzalkonium chloride; and or-   (8) buffers; and or-   (9) Diluents such as pharmaceutically acceptable inert fillers, such    as microcrystalline cellulose, lactose, dibasic calcium phosphate,    saccharides, and/or mixtures of any of the foregoing; and or-   (10) wetting agents such as corn starch, potato starch, maize    starch, and modified starches, croscarmellose sodium, crosspovidone,    sodium starch glycolate, and mixtures thereof; and or-   (11) disintegrants; and or-   (12) effervescent agents such as effervescent couples such as an    organic acid (e.g., citric, tartaric, malic, fumaric, adipic,    succinic, and alginic acids and anhydrides and acid salts), or a    carbonate (e.g. sodium carbonate, potassium carbonate, magnesium    carbonate, sodium glycine carbonate, L-lysine carbonate, and    arginine carbonate) or bicarbonate (e.g. sodium bicarbonate or    potassium bicarbonate); and or-   (13) other pharmaceutically acceptable excipients.

Pharmaceutical compositions suitable for use in animals and inparticular in man typically must be sterile and stable under theconditions of manufacture and storage. The pharmaceutical compositioncomprising nanoparticles can be formulated as a solution, microemulsion,liposome, or other ordered structure suitable to high drugconcentration.

Pharmaceutical compositions of the invention can be administered tohumans and animals in any pharmaceutically acceptable manner, such asorally, rectally, pulmonary, intravaginally, locally (powders, ointmentsor drops), transdermal, or as a buccal or nasal spray.

Raloxifene is subject to significant first-pass metabolism, whichimpacts on bioavailability. Conventionally formulated raloxifene isgenerally considered not to be amenable to transdermal delivery.However, the particulate raloxifene, or pharmaceutically acceptable saltor solvate of the present invention is more amenable to such delivery.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, pellets, granules, and the like. Such dosage forms mayalso comprise buffering agents.

Pharmaceutical compositions of the invention may be parenterallyadministered as a solution of the particulate raloxifene hydrochloridesuspended in an acceptable carrier, preferably an aqueous carrier. Avariety of aqueous carriers may be used, e.g., water, buffered water,0.4% saline, 0.3% glycine, hyaluronic acid and the like. Thesecompositions may be sterilized by conventional, well known sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile solution prior toadministration.

For aerosol administration, pharmaceutical compositions of the inventionare preferably supplied along with a surface stabilizer and propellant.The surface stabilizer must be non-toxic, and preferably soluble in thepropellant. Representative of such agents are the esters or partialesters of fatty acids containing from 6 to 22 carbon atoms, such ascaproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic,olesteric and oleic acids with an aliphatic polyhydric alcohol or itscyclic anhydride. Mixed esters, such as mixed or natural glycerides maybe employed. The surface stabilizer may constitute 0.1%-20% by weight ofthe composition, preferably 0.25-5%. The balance of the composition isordinarily propellant. A carrier can also be included, as desired, aswith, e.g., lecithin for intranasal delivery.

Pharmaceutical compositions of the invention may also be administeredvia liposomes, which serve to target the active agent to a particulartissue, such as lymphoid tissue, or targeted selectively to cells.Liposomes include emulsions, foams, micelles, insoluble monolayers,liquid crystals, phospholipid dispersions, lamellar layers and the like.In these preparations the nanocomposite microstructure composition isincorporated as part of a liposome, alone or in conjunction with amolecule that binds to or with other therapeutic or immunogeniccompositions.

Additionally, the compounds of this invention are well suited toformulation as sustained release dosage forms. The formulations can alsobe so constituted that they release the active ingredient only orpreferably in a particular part of the intestinal tract, and/or over aperiod of time. Such formulations may include coatings, envelopes, orprotective matrices which may be made from polymeric substances orwaxes.

Suitable surface stabilisers may include CTAB, cetyl pyridiniumchloride, gelatin, casein, phosphatides, dextran, glycerol, gum acacia,cholesterol, tragacanth, stearic acid, stearic acid esters and salts,calcium stearate, glycerol monostearate, cetostearyl alcohol,cetomacrogol emulsifying wax, sorbitan esters, polyoxyethylene alkylethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitanfatty acid esters, polyethylene glycols, dodecyl trimethyl ammoniumbromide, polyoxyethylene stearates, colloidal silicon dioxide,phosphates, sodium dodecylsulfate, carboxymethylcellulose calcium,hydroxypropyl celluloses, hydroxypropyl methylcellulose,carboxymethylcellulose sodium, methylcellulose, hydroxyethylcellulose,hydroxypropylmethyl-cellulose phthalate, noncrystalline cellulose,magnesium aluminum silicate, triethanolamine, polyvinyl alcohol,polyvinylpyrrolidone, 4-(1,1,3,3-tetramethylbutyl)-phenol polymer withethylene oxide and formaldehyde, poloxamers, poloxamines, a chargedphospholipid, dimyristoyl phophatidyl glycerol, dioctylsulfosuccinate,dialkylesters of sodium sulfosuccinic acid, dioctyl sodiumsulfosuccinate, sodium lauryl sulfate, alkyl aryl polyether sulfonates,mixtures of sucrose stearate and sucrose distearate, triblock copolymersof the structure: -(-PEO)-(-PBO-)-(-PEO-)-,p-isononylphenoxypoly-(glycidol), decanoyl-N-methylglucamide; n-decylβ-D-glucopyranoside, n-decyl β-D-maltopyranoside, n-dodecylβ-D-glucopyranoside, n-dodecyl β-D-maltoside,heptanoyl-N-methylglucamide, n-heptyl-β-D-glucopyranoside, n-heptylβ-D-thioglucoside, n-hexyl β-D-glucopyranoside,nonanoyl-N-methylglucamide, n-noyl β-D-glucopyranoside,octanoyl-N-methylglucamide, n-octyl-β-D-glucopyranoside, octylβ-D-thioglucopyranoside, lysozyme, a PEG derivatized phospholipid, PEGderivatized cholesterol, a PEG derivatized cholesterol derivative, PEGderivatized vitamin A, PEG derivatized vitamin E, and random copolymersof vinyl acetate and vinyl pyrrolidone, and/or mixtures of any of theforegoing.

Suitable surface stabilisers may also include a cationic surfacestabilizer selected from the group consisting of a polymer, abiopolymer, a polysaccharide, a cellulosic, an alginate, a nonpolymericcompound, and a phospholipid.

Suitable surface stabilisers may also include a surface stabilizerselected from the group consisting of cationic lipids, benzalkoniumchloride, sulfonium compounds, phosphonium compounds, quarternaryammonium compounds, benzyl-di(2-chloroethyl)ethylammonium bromide,coconut trimethyl ammonium chloride, coconut trimethyl ammonium bromide,coconut methyl dihydroxyethyl ammonium chloride, coconut methyldihydroxyethyl ammonium bromide, decyl triethyl ammonium chloride, decyldimethyl hydroxyethyl ammonium chloride, decyl dimethyl hydroxyethylammonium chloride bromide, C₁₂₋₁₅dimethyl hydroxyethyl ammoniumchloride, C₁₂₋₁₅dimethyl hydroxyethyl ammonium chloride bromide, coconutdimethyl hydroxyethyl ammonium chloride, coconut dimethyl hydroxyethylammonium bromide, myristyl trimethyl ammonium methyl sulphate, lauryldimethyl benzyl ammonium chloride, lauryl dimethyl benzyl ammoniumbromide, lauryl dimethyl (ethenoxy)₄ ammonium chloride, lauryl dimethyl(ethenoxy)₄ ammonium bromide, N-alkyl (C₁₂₋₁₈)dimethylbenzyl ammoniumchloride, N-alkyl (C₁₄₋₁₈)dimethyl-benzyl ammonium chloride,N-tetradecylidmethylbenzyl ammonium chloride monohydrate, dimethyldidecyl ammonium chloride, N-alkyl and (C₁₂₋₁₄) dimethyl 1-napthylmethylammonium chloride, trimethylammonium halide, alkyl-trimethylammoniumsalts, dialkyl-dimethylammonium salts, lauryl trimethyl ammoniumchloride, ethoxylated alkyamidoalkyldialkylammonium salt, an ethoxylatedtrialkyl ammonium salt, dialkylbenzene dialkylammonium chloride,N-diclecyldimethyl ammonium chloride, N-tetradecyldimethylbenzylammonium, chloride monohydrate, N-alkyl(C₁₂₋₁₄) dimethyl1-naphthylmethyl ammonium chloride, dodecyldimethylbenzyl ammoniumchloride, dialkyl benzenealkyl ammonium chloride, lauryl trimethylammonium chloride, alkylbenzyl methyl ammonium chloride, alkyl benzyldimethyl ammonium bromide, C₁₂ trimethyl ammonium bromides, C₁₅trimethyl ammonium bromides, C₁₇ trimethyl ammonium bromides,dodecylbenzyl triethyl ammonium chloride, poly-diallyldimethylammoniumchloride (DADMAC), dimethyl ammonium chlorides, alkyldimethylammoniumhalogenides, tricetyl methyl ammonium chloride, decyltrimethylammoniumbromide, dodecyltriethylammonium bromide, tetradecyltrimethylammoniumbromide, methyl trioctylammonium chloride, POLYQUAT 10™,tetrabutylammonium bromide, benzyl trimethylammonium bromide, cholineesters, benzalkonium chloride, stearalkonium chloride compounds, cetylpyridinium bromide, cetyl pyridinium chloride, halide salts ofquaternized polyoxyethylalkylamines, MIRAPOL™, ALKAQUAT™, alkylpyridinium salts; amines, amine salts, amine oxides, imide azoliniumsalts, protonated quaternary acrylamides, methylated quaternarypolymers, cationic guar, polymethylmethacrylate trimethylammoniumbromide, polyvinylpyrrolidone-2-dimetbylaminoethyl methacrylate dimethylsulfate, hexadecyltrimethyl ammonium bromide, poly(2-methacryloxyethyltrimethylammonium bromide) (S1001),poly(N-vinylpyrrolidone/2-dimethylaminoethyl methacrylate) dimethylsulphate quarternary (S1002), (S-630)poly(pyrrolidone-co-vinylacetate) andpoly(2-methylacryloxyamidopropyltrimethylammonium chloride) (S1004).

In some embodiments the preferred surface stabilizer is CTAB.

In embodiments in which the particulate raloxifene hydrochloride isproduced using a method of the invention, and the method of theinvention utilises a surface stabilizer, in a preferred form of theinvention, the surface stabilizer of the pharmaceutical composition isthe same surface stabilizer as that used in the method. As would beunderstood by person skilled in the art, it may be desirable to addfurther quantities of the surface stabilizer to the particulateraloxifene hydrochloride for the purposes of preparing a pharmaceuticalcomposition.

In embodiments in which the particulate raloxifene, or pharmaceuticallyacceptable salt or solvate thereof, is produced using a method of theinvention, and the method of the invention utilises a grinding compound,in a preferred form of the invention, the water-soluble diluent of thepharmaceutical composition is the same as the grinding compound used inthe method. As would be understood by person skilled in the art, it maybe desirable to add further quantities of the water soluble diluent tothe particulate raloxifene hydrochloride for the purposes of preparing apharmaceutical composition, relative to the quantity of grindingcompound used in the method, or to remove some of the grinding compoundprior to preparation of the composition.

In one form, the pharmaceutical composition of the invention is an oraldosage form comprising particulate raloxifene according to theinvention, or pharmaceutically acceptable salt or solvate thereof,according to the invention, a surfactant in the form of CTAB, and awater-soluble diluent in the form of sodium chloride.

As a further embodiment of the invention, the particulate raloxifene, orpharmaceutically acceptable salt or solvate thereof, may be administeredalong with an effective amount of an additional therapeutic agent,including but not limited to estrogen, progestin, benzothiophenecompounds including raloxifene, naphthyl compounds having antiestrogenactivity, bisphosphonate compounds such as alendronate and tiludronate,parathyroid hormone (PTH), including truncated and/or recombinant formsof PTH such as, for example, PTH (1-34), calcitonin, bone morphogenicproteins (BMPs), or combinations thereof. The different forms of theseadditional therapeutic agents available as well as the various utilitiesassociated with same and the applicable dosing regimens are well knownto those of skill in the art.

Various forms of estrogen and progestin are commercially available. Asused herein, the term “estrogen” includes compounds having estrogenactivity and estrogen-based agents. Estrogen compounds useful in thepractice of the present invention include, for example, estradiolestrone, estriol, equilin, equilenin, estradiol cypionate, estradiolvalerate, ethynyl estradiol, polyestradiol phosphate, estropipate,diethylstibestrol, dienestrol, chlorotrianisene, and mixtures thereof.Estrogen-based agents, include, for example, 17-.alpha.-ethynylestradiol (0.01-0.03 mg/day), mestranol (05-0.15 mg/day), and conjugatedestrogenic hormones such as Premarin™ (Wyeth-Ayerst; 0.2-2.5 mg/day). Asused herein, the term “progestin” includes compounds havingprogestational activity such as, for example, progesterone,norethynodrel, norgestrel, megestrol acetate, norethindrone,progestin-based agents, and the like. Progestin-based agents include,for example, medroxyprogesterone such as Provera™ (Upjohn; 2.5-10mg/day), norethylnodrel (1.0-10.0 mg/day), and norethindrone (0.5-2.0mg/day). A preferred estrogen-based compound is Premarin™, andnorethylnodrel and norethindrone are preferred progestin-based agents.The method of administration of each estrogen- and progestin-based agentis consistent with that known in the art.

The present invention will now be described with reference to thefollowing non-limiting Examples. The description of the Examples is inno way limiting on the preceding paragraphs of this specification, butis provided for exemplification of the methods and compositions of theinvention.

Examples

It will be apparent to persons skilled in the materials andpharmaceutical arts that numerous enhancements and modifications can bemade to the above described processes without departing from the basicinventive concepts. For example, in some applications the biologicallyactive compound substrate may be pretreated and supplied to the processin the pretreated form. All such modifications and enhancements areconsidered to be within the scope of the present invention, the natureof which is to be determined from the foregoing description and theappended claims. Furthermore, the following Examples are provided forillustrative purposes only, and are not intended to limit the scope ofthe processes or compositions of the invention.

A. Processing of Diclofenac Acid with Sodium Chloride Grinding Compound

A mixture consisting of a biologically active compound in the form of0.439 g of diclofenac acid (DCA)

and grinding compound in the form of 3.681 g of sodium chloride (therebyproviding the mixture at 10.7 and 89.3 weight % respectively,corresponding to 15 and 85 volume %, with a total volume of 2 cm³) wasdry milled for 15 minutes using a Spex 8000D mixer/mill with a 70 cm³hardened steel ball mill container containing ten 10 mm (40 g) stainlesssteel balls as the milling media. This resulted in the formation of adispersion comprising DCA in nanoparticulate form dispersed within amatrix of grinding compound sodium chloride.

In order to examine the effect of volume ratio of DCA to NaCl onparticle size, milling experiments were carried out with: 5 vol % (3.43w %), 10 vol % (7 w %), 30 vol % (22.5 w %) and 50 vol % (45 w %) of DCAto NaCl (total volume of 2 cm³, 15 minute milling time).

Ultra-fine particles of diclofenac acid in nanoparticulate form wererecovered by removing the grinding compound through washing with dilutehydrochloric acid. The washed powder was subsequently dried at roomtemperature for several hours in air.

In order to remove the grinding compound from the diclofenac acid innanoparticulate form, the dispersion was washed as follows. To obtain0.25 g of diclofenac particles varying amounts of dispersion were used,depending on the volume percentage. For a 15 vol % DCA dispersion, 2.339g was slowly added to 40 mL of a vigorously stirred solution of 0.01 MHCl and 1 mM CTAB (cetyl trimethyl ammonium bromide) in a conical flask.The sample was stirred for 30 minutes and filled into 15 mL plastictubes for centrifugation (falcon tubes). The sample was then subjectedto 3 repeats of: centrifugation (whereas the centrifugation speed wasincreased for each washing step from 5,000 g to 8,000 g and finally to12,000 g for a period of 3 minutes), removal of supernatant, addition of0.01 M HCl and 1 mM CTAB, and redispersion by vortex and ultrasoundsonication (5-10 seconds each).

The SEM and TEM images (FIG. 4) demonstrate that nanoparticles of adiameter in the order of 100-200 nm size range after washing. BETresults illustrated in FIG. 1, show the highest surface area(11.755±0.1035 m²/g) obtained was for the 5 vol % DCA. As can be seenfrom FIG. 2, Dynamic light scatter (DLS) analysis showed particle sizesof 160±30 nm.

The resulting dispersion for the 15 vol % sample, stabilized with thesurface stabilizer CTAB, was found to comprise the diclofenac acid formof the drug (by XRD, FTIR and DSC), with nanoparticles less than 200 nmand the majority on the order of 30-50 nm. TEM of the DCA innanoparticulate form (after washing the dispersion and stabilized withthe surface stabilizer CTAB) also showed both spherical and nonsphericalnanoparticles, the nonspherical particles appearing to be rod-shaped,having a minor-axis dimension of about 30 nm and a major-axis dimensionof about 150 nm. DSC analysis of the melting point of the DCA innanoparticulate form confirmed its identity as diclofenac acid, with amelting point in the range of 175−185° C.

FIG. 7 illustrates the effect of increasing milling time of diclofenacacid with NaCl grinding compound, 15 vol %), showing that the meltingpoint shifts to lower temperatures, likely due to a decrease of thediameter of the particles of diclofenac acid.

B. Processing of Olanzapine Plus Sodium Chloride Grinding Compound

A biologically active compound in the form of 0.39 g of conventionalolanzapine powder,

was placed in a milling apparatus (a 70 cm³ stainless steel ball millcontainer) with grinding compound in the form of 3.68 g of NaCl, therebyproviding the mixture at 9.6 and 90.4 weight % respectively,corresponding to 15 and 85 volume %, with a total volume of 2 cm³.Milling media comprising 40 g of 10 mm steel balls (10 pieces) wereemployed in the container. The milling apparatus was closed under housevacuum prior to milling. Cooling was achieved with compressed air flow(100 kcpa). The mixture was dry milled for 15 minutes and 180 minutes,the composition resulting after milling for both times comprisedolanzapine in nanoparticulate form dispersed in the grinding mediumNaCl.

As can be seen from FIG. 3, scanning electron microscopy (SEM) of theresulting dispersion showed nanocrystalline structures and nanoparticlesof olanzapine on the order of 100 nm. Milling times assayed included 15and 180 minutes in two separate milling runs. Analysis of the meltingpoint of the nanoparticles produced at 180 minute milling time periodconfirmed that the resulting composition was olanzapine, with a meltingpoint in the range of 200° C. Material showed some decolourization at180 minutes, which is attributed to degradation of the drug.

C. Processing of Diclofenac Acid with Ammonium Chloride GrindingCompound

Biologically active compound in the form of 0.439 g of conventional DCApowder,

was placed in a milling apparatus (a 70 cm³ stainless steel ball millcontainer) with grinding compound in the form of 2.596 g of NH₄Cl,thereby providing the mixture at 14.5 and 85.5 weight % respectively,corresponding to 15 and 85 volume %, with a total volume of 2 cm³.Milling media comprising 40 g of 10 mm steel balls (10 pieces) wereemployed in the mill. Cooling was achieved with compressed air flow (100kcpa). Milling time was 15 minutes and the composition resulting aftermilling comprised DCA in nanoparticulate form dispersed in NH₄Clgrinding compound.

The nanoparticle size can be seen from a representative TEM afterwashing (FIG. 5) and is about 200 nm in diameter (washing with 0.01 MHCl and 1 mM CTAB was performed as described for DCA NaCl milling). Themelting point of DSC shows that the nanoparticles are obtained as thediclofenac acid (FIG. 6), The melting point of diclofenac acid is afterliterature at 182° C., one can see a melting point at 177° C., the shiftis probably due to the small particles size. The large peak at 194° C.is due to NH₄Cl.

D. Raloxifene

In embodiments in which the particulate raloxifene, or pharmaceuticallyacceptable salt or solvate thereof, is produced using a method of theinvention, and the method of the invention utilises a grinding compound,in a preferred form of the invention, the water-soluble diluent of thepharmaceutical composition is the same as the grinding compound used inthe method. As would be understood by person skilled in the art, it maybe desirable to add further quantities of the water soluble diluent tothe particulate raloxifene hydrochloride for the purposes of preparing apharmaceutical composition, relative to the quantity of grindingcompound used in the method, or to remove some of the grinding compoundprior to preparation of the composition.

In one form, the pharmaceutical composition of the invention is an oraldosage form comprising particulate raloxifene according to theinvention, or pharmaceutically acceptable salt or solvate thereof,according to the invention, a surfactant in the form of CTAB, and awater-soluble diluent in the form of sodium chloride.

As a further embodiment of the invention, the particulate raloxifene, orpharmaceutically acceptable salt or solvate thereof, may be administeredalong with an effective amount of an additional therapeutic agent,including but not limited to estrogen, progestin, benzothiophenecompounds including raloxifene, naphthyl compounds having antiestrogenactivity, bisphosphonate compounds such as alendronate and tiludronate,parathyroid hormone (PTH), including truncated and/or recombinant formsof PTH such as, for example, PTH (1-34), calcitonin, bone morphogenicproteins (BMPs), or combinations thereof. The different forms of theseadditional therapeutic agents available as well as the various utilitiesassociated with same and the applicable dosing regimens are well knownto those of skill in the art.

Various forms of estrogen and progestin are commercially available. Asused herein, the term “estrogen” includes compounds having estrogenactivity and estrogen-based agents. Estrogen compounds useful in thepractice of the present invention include, for example, estradiolestrone, estriol, equilin, equilenin, estradiol cypionate, estradiolvalerate, ethynyl estradiol, polyestradiol phosphate, estropipate,diethylstibestrol, dienestrol, chlorotrianisene, and mixtures thereof.Estrogen-based agents, include, for example, 17-.alpha.-ethynylestradiol (0.01-0.03 mg/day), mestranol (05-0.15 mg/day), and conjugatedestrogenic hormones such as Premarin™ (Wyeth-Ayerst; 0.2-2.5 mg/day). Asused herein, the term “progestin” includes compounds havingprogestational activity such as, for example, progesterone,norethynodrel, norgestrel, megestrol acetate, norethindrone,progestin-based agents, and the like. Progestin-based agents include,for example, medroxyprogesterone such as Provera™ (Upjohn; 2.5-10mg/day), norethylnodrel (1.0-10.0 mg/day), and norethindrone (0.5-2.0mg/day). A preferred estrogen-based compound is Premarin™, andnorethylnodrel and norethindrone are preferred progestin-based agents.The method of administration of each estrogen- and progestin-based agentis consistent with that known in the art.

Use for Alleviating Pathologies

As stated in the summary, the present invention further provides the useof the said particulate raloxifene, or pharmaceutically acceptable saltor solvate thereof, in the manufacture of a medicament for alleviatingpathologies, including osteoporosis, serum lipid lowering, andinhibiting endometriosis, uterine fibrosis, and breast cancer, and theuse of compositions comprising or formulated using the said particulateraloxifene, or pharmaceutically acceptable salt or solvate thereof, foralleviating pathologies, including osteoporosis, serum lipid lowering,and inhibiting endometriosis, uterine fibrosis, and breast cancer.

The present invention provides a method for the treatment of apathology, such as osteoporosis, serum lipid lowering, and inhibitingendometriosis, uterine fibrosis, and breast cancer by administration ofa therapeutically effective amount of particulate raloxifene, orpharmaceutically acceptable salt or solvate thereof, according to theinvention.

The particular dosage of particulate raloxifene, or pharmaceuticallyacceptable salt or solvate thereof, required to treat, inhibit, orprevent the symptoms and/or disease of a mammal, including humans,suffering from the above maladies according to this invention willdepend upon the particular disease, symptoms, and severity, as well asthe potential increased efficacy due to particulate form of theraloxifene, or pharmaceutically acceptable salt or solvate thereof,(e.g., increased solubility, more rapid dissolution, increased surfacearea). Amounts effective for such a use will depend on: the desiredtherapeutic effect; the route of administration; the potency of thetherapeutically active agent; the desired duration of treatment; thestage and severity of the disease being treated; the weight and generalstate of health of the patient; and the judgment of the prescribingphysician.

Generally, accepted and effective doses will be from 15 mg to 1000 mg,and more typically from 15 mg to 80 mg. Such dosages will beadministered to a patient in need of treatment from one to three timeseach day or as often as needed for efficacy, generally for periods of atleast two months, more typically for at least six months, orchronically.

As discussed above, the particulate raloxifene, or pharmaceuticallyacceptable salt or solvate thereof, of this invention can beadministered by a variety of routes, the selection of which will bedecided by the attending physician.

The raloxifene compounds of the current invention may be made accordingto established procedures, such as those detailed in U.S. Pat. Nos.4,133,814, 4,418,068, and 4,380,635, and European Patent Application95306050.6, Publication No. 0699672, Kjell, et al., filed Aug. 30, 1995,published Mar. 6, 1996, all of which are herein incorporated byreference. In addition, the information disclosed in the publishedEuropean Patent Application number 0670162 A1, published on Sep. 6,1995, is herein incorporated by reference.

Methods for the preparation of amorphous raloxifene salts and specificpharmaceutically acceptable salts are discussed earlier in thisspecification.

D1. Raloxifene HCl

Conventional active pharmaceutical compound raloxifene hydrochloride(0.5805 g) was introduced with NaCl (5.5208 g) into a steel vessel (75cm³) with milling bodies comprising 10×10 mm steel balls. The totalvolume of the raloxifene hydrochloride/salt mixture was 3 cm³ with 15vol % of drug. Both the raloxifene hydrochloride and the sodium chloridegrinding compound were kept dry prior to milling by storage under vacuumand over P₂O₅. The steel milling chamber was closed under vacuum toremove moisture and air, to reduce degradation/oxidization.

The milling chamber was mounted on a Spex ball mill and was shaken for15 min and cooled by a stream of compressed air. This resulted in theformation of a solid-dispersion consisting of raloxifene hydrochloridedispersed within a matrix of fine NaCl.

The milling chamber was then carefully opened to release the vacuum, andclosed to allow any airborne particles to settle. The milling chamberwas then opened in a fume hood to prevent inhalation of the fineparticles, and the contents transferred through a 2 mm sieve (to removethe milling bodies) into 8 mL glass vials and stored in a vacuumdesiccator over P₂O₅.

To remove the sodium chloride from the milled raloxifene hydrochloride,the solid-dispersion was washed as follows. The solid dispersion wasmixed with 0.1 g of the surfactant CTAB, and placed in a 25 mL Schottbottle. 20 mL of ice cooled solution of 0.1 M HCl and 1 mM cetyltrimethyl ammonium bromide (CTAB) were added. The bottle was closed andimmediately mounted in the Spex ball mill and shaken for 3 minutes.After the shaking procedure a pale yellow dispersion formed, and storedin an ice bath prior to centrifugation. The sample was then subjected tothe following (3×): centrifugation (the centrifugation speed wasincreased for each washing step from 6000 g to 8000 g and finally to12000 g for a period of 3 minutes each), removal of supernatant,addition of 0.01 M HCl and 1 mM CTAB, and redispersion by vortex mixing.

The dispersion was then transferred onto a watch glass and dried over astream of air. After drying for about 3 hours the suspension dried downto form a dry layer on the glass surface, this was stored over night ina vacuum desiccator over P₂O₅. This yielded 0.48 g of dried powder whichwas stored in a glass vial in a vacuum desiccator.

The dissolution properties of the particulate raloxifene hydrochloridewere tested with a USP apparatus in simulated gastric conditions, andcompared with commercial raloxifene hydrochloride. About 60 mg ofparticulate raloxifene hydrochloride and commercial raloxifenehydrochloride, respectively, were introduced into gelatin capsules. Thedissolution properties were followed as a function of solutionconcentration versus time.

The dissolution conditions were as follows: 1 L of 0.1 M HCl containing2 g of NaCl were degassed and brought to 37° C. in a USP conformdissolution vessel to paddle and stirred at about 80 rpm. The raloxifenehydrochloride was either tested as powder or in gelatin capsule with ametal sinker. For each time point, 2 mL of sample were removed from thesolution, to remove larger aggregates it was centrifuged for 1 min at10000 g and 1.5 ml was taken from the top of the solution and theconcentration was measured using a Waters HPLC running a methodvalidated with respect to specificity, linearity, precision andrepeatability.

The dissolution profile in FIG. 8 shows significantly enhancedsolubility properties of the particulate raloxifene hydrochloride asopposed to the commercial raloxifene hydrochloride, this can be seen forexample in a nearly five fold increase in solution concentration after50 minutes in simulated intestinal fluid. To understand these data itneeds to be emphasized that the conditions were kept close to themarketed dosage of raloxifene and the concentration of 60 mg of drug perliter are well above the solubility.

As can be seen from the scanning electron micrographs in FIG. 9a , thecommercial raloxifene HCl particles seem to have a broad sizedistribution with glassy particles of up to several micrometers. Aftersalt milling of raloxifene HCl with NaCl, small structures in the sizeof about 100-200 nm are a predominant feature (FIG. 9b ). After thewashing procedure and drying, small structures can be attributed toraloxifene HCl particles (as the salt matrix was removed by washing),the particle-like structures show a size of about 100-200 nm (FIGS. 9cand d ).

The size determined by SEM is in good agreement with the sizedistribution of the dispersion before drying by dynamic light scatter(DLS) (FIG. 10). Here the particles size was determined with a MalvernHPPS dynamic light scattering apparatus with a size distribution afternumber of 128±53 nm (number weighted) in intensity a second peak of 300nm was detected. Prior to the measurement any larger aggregates oragglomerates were removed by 1 min centrifugation at 6000 g, and onlythe supernatant was analyzed. This indicates that the particles obtainedin the milling process did not significantly grow during the washingprocedure.

Further evidence for the decreased particles size after milling andwashing are the BET surface area which increased from 0.1 m²/g for thecommercial raloxifene HCl to about 7 to 20 m²/g for the particulateraloxifene hydrochloride, which can be explained by the increasedsurface to mass ratio as the particles' diameter decreases. Surfaceareas for various examples are tabulated below.

sample No. BET surface area 169AW 15.6517 ± 0.0332 m²/g 169BW 17.0576 ±0.0368 m²/g 171AW 16.6507 ± 0.0297 m²/g 171BW 15.0311 ± 0.0244 m²/g172AW 18.7621 ± 0.0373 m²/g 172BW 21.1382 ± 0.0323 m²/g 174AW 13.9266 ±0.0320 m²/g 174BW 17.9952 ± 0.0317 m²/g 175AW 16.7377 ± 0.0199 m²/g175BW 25.1960 ± 0.0502 m²/g 176AW 20.6579 ± 0.0400 m²/g 176BW 11.9100 ±0.0221 m²/g 3011AW 10.1126 ± 0.0295 m²/g 3013BW  7.0471 ± 0.0202 m²/g3016AW  9.1337 ± 0.0394 m²/g 3011BW  10.505 ± 0.0290 m²/g 1177AW  9.1774± 0.0435 m²/g 3016BW (samples combined) 3017BW 3013AW 177B15

The melting point shows a ten degree Celsius reduced onset fornanoparticulate raloxifene HCl, as compared to the commercial product,being further evidence for a reduced particle size (FIG. 11).

The XRD-spectra (FIG. 12) shows that the nanoparticulate raloxifene HClappears to be in the same crystalline phase as the commercial raloxifeneHCl, and suggest that the particles remain crystalline. The relativebroadening of the peaks of the nanoparticles raloxifene as compared tothe commercial raloxifene is a further indicator of the reducedparticles size.

The solution ¹H-NMR-spectra, shown in FIG. 13, confirms that thecompound is identical to the commercial raloxifene HCl, it was alsodetermined that about 2 w % of the surfactant CTAB are present afterwashing and drying. The solution ¹H NMR spectra were measured of about10 mg of particulate raloxifene HCl and commercial raloxifene HCl (datanot shown) dissolved in d6-DMSO.

The FT-IR-spectra (FIG. 14) shows further the chemical identity ofraloxifene HCl and the nanoparticulate raloxifene HCl.

The salt content after washing was negligible, and as determined by ICPmeasurements, only about 0.65 w % of NaCl remained, while the commercialsample showed with about 0.08 w % NaCl not significantly lower saltconcentrations. This observation was also supported by the disappearanceof the NaCl pattern in the XRD-spectra of the sample after washing ascompared to the sample directly after salt milling (data not shown).

The chemical identify of the raloxifene HCl was further confirmed by thesimilarity of the IR-spectra, which is nearly identical. This alsoconfirms that the amount of CTAB after washing is quite small (FIG. 14).Nevertheless the reversal of the Zeta-potential seems to indicate thatthe surfactant does play an important role as well.

D2. Raloxifene HCl (Amorphous)

Conventional active pharmaceutical compound raloxifene hydrochloride(0.3867 g) was introduced with NaCl (3.672 g) into a steel vessel (75cm³) with milling bodies comprising 10×10 mm steel balls. The totalvolume of the raloxifene hydrochloride/salt mixture was 2 cm³ with 15vol % of drug. Both the raloxifene hydrochloride and the sodium chloridegrinding compound were used without any additional drying step prior tothe milling. The steel milling chamber was closed under vacuum to removemoisture from the air, and to reduce degradation/oxidization.

The milling chamber was mounted on a Spex ball mill and was shaken for15 min and cooled by a stream of compressed air. This resulted in theformation of a solid-dispersion consisting of raloxifene hydrochloridedispersed within a matrix of fine NaCl.

The milling chamber was then carefully opened to release the vacuum, andclosed to allow any airborne particles to settle. The milling chamberwas then opened in a fume hood to prevent inhalation of the fineparticles, and the contents transferred through a 2 mm sieve (to removethe milling bodies) into 8 mL glass vials and stored in a vacuumdesiccator over P₂O₅.

To remove the sodium chloride from the milled raloxifene hydrochloride,the solid-dispersion was washed as follows. About 4.1 g of the soliddispersion was placed in a 25 mL Schott bottle, and added 20 mL of icecooled solution of 0.1 M HCl and 1 mM sodium dodecyl sulfate (SDS) wasadded. The bottle was closed and immediately mounted in the Spex ballmill and shaken for 1 min. After the shaking procedure a pale yellowdispersion was formed, and stored in an ice bath prior tocentrifugation. The sample was then subjected to a centrifugation stepat 6000 g for a period of 3 minutes, and the supernatant was removed.The sample was dispersed with 4 mL of 0.1 M HCl and 1 mM SDS solution.

The dispersion was then transferred onto a watch glass and dried over astream of air. After drying for about 3 hours the suspension dried downto form a dry layer on the glass surface, this was stored over night ina vacuum desiccator over P₂O₅.

The XRD shows that after milling and washing the crystal structure ofraloxifene HCl is lost, and the broad increase in intensity from 10 to35 (2 Theta) is indicative for an amorphous phase (FIG. 15). The XRDspectrum shows the different processing stage, before milling, aftermilling, and after washing. The commercial Raloxifene HCl shows distinctpeaks that are due to its crystalline state.

Prevalent peaks after salt milling are mostly due to sodium chloride andthe usage of an aluminium sample holder, the peaks of raloxifene HCl cannot be identified, as they are too dilute in the matrix. After the onewashing step some peaks of the sodium chloride remain, but only a broadband of raloxifene HCl can be seen, which can be attributed to amorphousphase.

There are only a few peaks related to raloxifene HCl crystals pertained,indicating that some crystal order is formed. The XRD spectra also showssome that there is still some sodium chloride remaining in the sample,as can be seen from the peaks at about 27(2 Theta).

The SEM shows that some small particles were formed with a size of about100-200 nm. Some of the particles seem to be slightly elongated (FIG.16).

The BET surface area was measured to be 10.6 m²/g, which confirms that amaterial with high surface was formed and supports results from the SEMimage.

The IR-spectra, in particular the peak at 2960 cm⁻¹, indicates thatraloxifene HCl salt is present in the in both the milled and milled andwashed samples (FIG. 17). Some peaks are less pronounced then in thepure raloxifene HCl spectra, but this might be due to the remainingsalt.

D3. Raloxifene Free Base

0.3640 g Raloxifene free base and 3.6725 g NaCl (20 vol % drug) wasmilled with 10 pieces of 10 mm steel balls for 15 min at roomtemperature.

The SEM of the starting shows large pieces of glassy looking raloxifenebase, and has no fine structure, but is very smooth (FIG. 18 a and b).The SEM after salt milling shows in contrast a fine structure with smallparticles of about 100 nm in diameter that form larger agglomerates(FIG. 18 c and d). The particles are looking uniform in shape and nodifference between salt or drug can be observed.

After ball milling the salt was largely removed by dispersion in abuffer of pH 9, at which the solubility of raloxifene is very low, butNaCl would dissolve (0.01 M TRIS-Buffer, pH adjusted with HCl) (TRIS:(tris(hydroxymethyl)aminomethane hydrochloride). To colloidallystabilize the particles and to prevent aggregation the nonionicsurfactant Plasdone® S-630 (0.5 g/mL) and the ionic surfactant sodiumdodecyl sulphate SDS (0.2 mM) were added (FIG. 19). The dispersion wasstirred in 50 mL of the before mentioned mixture with a magnetic stirrerbar for 15 min, followed by a few seconds of ultrasonication in a waterbath.

The dispersion was then washed by centrifugation using 15 mL Falcontubes and a centrifugation speed of 5,000 g. The supernatant wasdiscarded and the sediment was dispersed with 30 mL of 0.01 MTris-buffer (pH 9) and 1 mM SDS by shaking. A further centrifugation at5,000 g followed and the pellet was dispersed with 3 mL of 0.01M Tris(pH 9) and 1 mM SDS.

The dispersion was dried over a nitrogen stream and the vacuum dried.The SEM image in FIG. 20 reveals a fine structure on the nanoscale,which shows structures of under 100 nm. The particles seem to have driedin a network like structure, probably bridged by the polymericsurfactant Plasdone.

The BET surface area indicated a very large surface area of57.7178±0.4095 m²/g. Calculating with a density of 1.3 g/cm³, andassuming monodispersed nanoparticles, the particles diameter with such asurface area would be about 80 nm. (For a density of 1.2 g/cm³: 85 nmand for 1.4 g/cm³, 70 nm).

The XRD shows that the raloxifene base is amorphous and the resultingproduct after washing is likewise amorphous, it also shows thedistinctly different peaks to the raloxifene HCl salt (FIG. 21). Thepowder XRD shows that raloxifene (ralox) free base is retained aftermilling and washing. The spectra of the salt milling sample is dominatedby the strong peak of NaCl; after washing the lower intensity of theNaCl peaks shows that the amount of NaCl is greatly reduced and revealsthe amorphous phase of raloxifene free base. The raloxifene free baseseems to contain still a small amount of NaCl, as compared to the sampledirectly after salt milling before the salt matrix was removed.

By diffuse reflection IR it was shown that the free base is retainedafter ball milling and after dissolution of the matrix (FIG. 22), as thepeak at about 2900 cm⁻¹ indicates. After dissolution of the matrix theadditional peak at about 1750 cm⁻¹ is most probably due to the non-ionicsurfactant Plasdone S-630, which probably covers the surface of thenanoparticles.

It can be concluded that sub 100 nm particles of amorphous raloxifenefree base, coated with the non-ionic surfactant Plasdone can be formed.

D4. Animal Studies

This study involved the investigation of the pharmacokinetics ofraloxifene hydrochloride following oral administration of two dosageformulations to 12 male and female beagle dogs. The two dosage formsinvestigated were 1) raloxifene hydrochloride particles, developedaccording to the method of the present invention, and 2) standard API.Both forms were administered as capsules prepared by the Pharmaceuticslaboratory of TetraQ.

As tested prior to administration to the dogs, the particulateraloxifene hydrochloride had the following properties: >75% of theparticles were in the range of 220-350 nm in size, with >90% in therange of 160-342 nm. Differential scanning calorimetry, or DSC, showedan approximate 10° C. reduction in the onset of melting as compared tothe comparison API. Dissolution of the particulate raloxifene understandard conditions in simulated gastric fluid and simulated intestinalfluid (60 mg in 1 L fluid) showed a significant increase in dissolutionas compared to the comparison API. At 90 minutes post addition,approximately 15 mg/L of particulate raloxifene was detected versus 8mg/L of comparison API for SGF; and 4.5 mg/L versus 0.75 mg/L for SIF.

Note that a less significant increase was observed when samples had notbeen subjected to brief grinding using a mortar and pestle. Grinding hadno effect on the dissolution profile of the comparison API, howeverincreased both dissolution rate and solubility for the raloxifenehydrochloride. This is consistent with the presence of looseagglomerates in the particulate raloxifene, but not the comparison API.

The study was designed as a crossover trial conducted in 6 male and 6female healthy Beagle dogs. Raloxifene was administered as an oral dosewith three male plus three female dogs each receiving one of the twopreparations on each of two dosing occasions. Eleven plasma samples werecollected from each animal in the 24 hour period following each dose andthese were all available for determination of raloxifene concentration.

Plasma samples were transported to the TetraQ-ADME laboratories on dryice and all were in an intact (frozen) condition upon arrival. Theconcentration of raloxifene in the plasma samples was determined by anLC-MS/MS assay developed and validated by TetraQ-ADME.

Pharmacokinetic analysis was performed using purpose-written macros forExcel Software. Standard model-independent pharmacokinetic methods wereused. Nominal sampling times were used in the calculations. The plasmaraloxifene concentrations were used to determine the followingparameters:

(i) C_(max) Maximum plasma concentration, read directly from the rawdata. (ii) T_(max) Time at which C_(max) was achieved, also readdirectly from the raw data. (iii) k_(e) Terminal elimination rateconstant, which was determined as the slope of the regression line ofbest fit to the approximately log-linear terminal elimination phase(using the least squares linear regression function in the Excel 2003software). The data from the final three or four measurable concentra-tions were used in the regression analysis for all data sets. (iv)t_(1/2) Terminal elimination half-life = ln 2/k_(e) (v) AUC_(0-t) Areaunder the plasma concentration-time curve from time zero to the time ofthe last measurable raloxifene concentration above the lower limit ofquantitation of the assay, determined by trapezoidal rule integration.Concentration values less than the lower limit of quantification (LLOQ)which occurred prior to the first measurable concentration were set tozero. (vi) AUC_(t-∞) Area under the plasma-concentration-time curve fromthe time of the last measured plasma concentration to infinity, asdetermined by the formula C_(t)/k_(e), where C_(t) is the concentrationvalue calculated on the line of best fit at the time when the lastmeasured plasma concentration occurred, and k_(e) is the terminalelimination rate constant as defined above. (vii) AUC_(0-∞) Sum ofAUC_(0-t) and AUC_(t-∞).

The individual subject data have been plotted using linear concentrationscales and are presented in FIG. 23. The iCeutica raloxifene HClnanoparticles are generally labelled as Test Substance 1 and thecommercial available raloxifene HCl is labelled as Test Substance 2. Themean±SD values at each nominal sampling time for each dosing group areshown in tabular and graphical form in FIG. 24.

Mean±SD data for each dosing group are displayed in FIG. 25. Anadditional comparison is made for C_(max) and AUC_(0-t) results for thetwo dosing groups in FIG. 26.

Administration of the particulate raloxifene HCl resulted in anapproximate 59% increase in maximum plasma concentration (C_(max)) and56% increase in area under the concentration vs. time curve (AUC_(0-t))compared with those following administration of the commercial API(12.26±5.47 and 7.69±4.54 ng/mL, and 33.39±20.54 and 21.36±16.79ng·h/mL, respectively). In addition, the median time to maximumconcentration (T_(max)) was shorter (1.00 vs. 1.50 hours, respectively)following the administration of the particulate raloxifene HCl comparedwith that following administration of the commercial API.

Analysis of AUC_(0-t) and C_(max) data for individual animals showed allto follow a pattern of higher results following administration of theparticulate raloxifene HCl compared to the commercial API, except thosefor one female animal, for which lower results were obtained forAUC_(0-t) and C_(max) following administration of the particulateraloxifene HCl. The results for this dog were clearly inconsistent withthose for the other animals; however no explanation can be offered forthis apparent inconsistency in results.

The variation between results obtained was reasonably high followingdosing of both formulations. No formal statistical comparison of thedata has been performed. However, the higher C_(max) and AUC_(0-t)results, as well as shorter T_(max) results, suggests that theformulations of the present invention have the potential to influencethe plasma pharmacokinetics of raloxifene in a manner which will resultin higher plasma concentrations being achieved both initially andthroughout the treatment period.

E. Processing of Fenofibrate with Sodium Chloride Grinding Compound in aCooled Attrition Mill

Biologically active compound in the form of 5 g of fenofibrate,

was placed in a 110 mL attrition mill (Union Process, Modified Model01-HD) with 37 g sodium chloride corresponding to a 1:7 volume ratio(15%:85%) in a 20 mL volume with approximately 1 kg of 0.25 inchstainless steel milling balls. The milling vessel was maintained at 0°C. through use of an external circulating chillier and milling wasconducted under an argon gas flow. Milling was conducted at 500 rpm for30, 45 and 60 minutes and the particles were washed in in deionizedwater to remove sodium chloride. FIG. 27 shows SEM pictures illustratingresultant particles of approximately 700 nm, 500 nm and less than 50 nm.

F. Raloxifene HCl Milled Using a Lactose Grinding Compound

Conventional active pharmaceutical compound raloxifene hydrochloride(0.5805 g) was introduced with lactose (4.284 g) into a steel vessel (75cm³) with milling bodies comprising 10×10 mm steel balls. The totalvolume of the raloxifene hydrochloride/lactose mixture was 3 cm³ with 15vol % of drug. Both the raloxifene hydrochloride and the lactosegrinding compound were kept dry prior to milling by storage under vacuumand over P₂O₅. The steel milling chamber was closed under vacuum toremove moisture and air, to reduce degradation/oxidization.

The milling chamber was mounted on a Spex ball mill and was shaken for15 min and cooled by a stream of compressed air. This resulted in theformation of a solid-dispersion consisting of raloxifene hydrochloridedispersed within a matrix of lactose.

The milling chamber was then carefully opened to release the vacuum, andany airborne particles allowed to settle. The milling chamber was openedin a fume hood to prevent inhalation of the fine particles, and thecontents transferred through a 2 mm sieve (to remove the milling bodies)into 8 mL glass vials and stored in a vacuum desiccator over P₂O₅.

As can be seen from the SEM images (FIG. 28), the mixture of drug andlactose grinding compound contain particles below 100 nm which arebelieved to represent the raloxifene drug. FIG. 28a shows that somesmall particles well below 5 micron meters are formed after milling. Athigher magnification a substructure, that shows particles with nanoparticle elements of about 100 nm can be seen

The dissolution of the particles in lactose and NaCl grinding compoundswere compared to the commercial API (FIG. 29). Both lactose and NaClmilled raloxifene showed enhanced dissolution properties relative to theAPI, which illustrates the ability to enhance the dissolution propertiesof drugs using a variety of grinding compounds and also demonstratesthat the grinding compound need not be separated from the API prior toformulation to retain those enhanced properties.

G. Olanzapine Free Base Milled Using a Lactose Grinding Compound

Conventional active pharmaceutical compound olanzapine free base (0.5846g) was introduced with lactose (4.284 g) into a steel vessel (75 cm³)with milling bodies comprising 10×10 mm steel balls. The total volume ofthe olanzapine/lactose mixture was 3 cm³ with 15 vol % of drug. Both theolanzapine freebase and the lactose grinding compound were kept dryprior to milling by storage under vacuum and over P₂O₅. The steelmilling chamber was closed under vacuum to remove moisture and air, toreduce degradation/oxidization.

The milling chamber was mounted on a Spex ball mill and was shaken for15 min and cooled by a stream of compressed air. This resulted in theformation of a solid-dispersion consisting of olanzapine free basedispersed within the lactose grinding compound.

The milling chamber was then carefully opened to release the vacuum, andclosed to allow any airborne particles to settle. The milling chamberwas then opened in a fume hood to prevent inhalation of the fineparticles, and the contents transferred through a 2 mm sieve (to removethe milling bodies) into 8 mL glass vials and stored in a vacuumdesiccator over P₂O₅.

As can be seen from FIG. 30, fine particles can be obtained aftermilling in a lactose grinding compound.

It is expected that a broad range of GRAS compounds can be used as agrinding compound for the purposes of the present invention. However,some grinding compounds may offer specific advantages. For example,olanzapine-lactose grinding compound mixtures produced under similarconditions to olanzapine-sodium chloride grinding compound mixturesappear to exhibit superior flowability, which is advantageous inautomated formulation systems.

It will be apparent to persons skilled in the materials andpharmaceutical arts that numerous enhancements and modifications can bemade to the above described processes without departing from the basicinventive concepts. For example, in some applications the precursorbiologically active agent compound may be pretreated and supplied to theprocess in the pretreated form. All such modifications and enhancementsare considered to be within the scope of the present invention, thenature of which is to be determined from the foregoing description andthe appended claims. Furthermore, the preceding examples are providedfor illustrative purposes only, and are not intended to limit the scopeof the processes or compositions of the invention.

We claim:
 1. A method for producing a composition comprisingnanoparticles of a biologically active compound, comprising the step of:dry milling a solid biologically active compound and a millable grindingcompound in a mill comprising a plurality of milling bodies, for a timeperiod sufficient to produce a solid dispersion comprising nanoparticlesof the biologically active compound dispersed in an at least partiallymilled grinding compound.
 2. The method of claim 1, wherein thenanoparticles have an average size less than a size selected from thegroup consisting of 200 nm, 100 nm, 75 nm, 50 nm, and 40 nm.
 3. Themethod of claim 2, wherein the particle size of at least 50% of thenanoparticles is within the average size range.
 4. The method of claim3, wherein the particle size of at least 75% of the nanoparticles iswithin the average size range.
 5. The method of any preceding claim,wherein the time period is a range selected from the group consisting ofbetween 5 minutes and 2 hours, between 5 minutes and I hour, between 5minutes and 45 minutes, between 5 minutes and 30 minutes, and between 10minutes and 25 minutes.
 6. The method of any of claims 1-5, wherein themilling medium is selected from the group consisting of ceramics,glasses, polymers, ferromagnetics, and metals.
 7. The method of claim 6,wherein the milling medium is steel balls having a diameter selectedfrom the group consisting of between 1 and 20 mm, between 2 and 15 mm,and between 3 and 10 mm.
 8. The method of any preceding claim whereinthe biologically active compound is selected from the group consistingof biologics, amino acids, proteins, peptides, nucleotides, nucleicacids, and analogs, homologs and first order derivatives.
 9. The methodof claim 8, wherein the biologically active compound is selected fromthe group consisting of anti-obesity drugs, central nervous systemstimulants, carotenoids, corticosteroids, elastase inhibitors,anti-fungals, oncology therapies, anti-emetics, analgesics,cardiovascular agents, anti-inflammatory agents, such as NSAIDs andCOX-2 inhibitors, anthelmintics, anti-arrhythmic agents, antibiotics(including penicillins), anticoagulants, antidepressants, antidiabeticagents, antiepileptics, antihistamines, antihypertensive agents,antimuscarinic agents, antimycobacterial agents, antineoplastic agents,immunosuppressants, antithyroid agents, antiviral agents, anxiolytics,sedatives (hypnotics and neuroleptics), astringents, alpha-adrenergicreceptor blocking agents, beta-adrenoceptor blocking agents, bloodproducts and substitutes, cardiac inotropic agents, contrast media,cough suppressants (expectorants and mucolytics), diagnostic agents,diagnostic imaging agents, diuretics, dopaminergics (anti-parkinsonianagents), haemostatics, immunological agents, lipid regulating agents,muscle relaxants, parasympathomimetics, parathyroid calcitonin andbiphosphonates, prostaglandins, radio-pharmaceuticals, sex hormones(including steroids), anti-allergic agents, stimulants and anoretics,sympathomimetics, thyroid agents, vasodilators, and xanthines.
 10. Themethod of claim 9, wherein the biologically active compound is selectedfrom the group consisting of haloperidol, DL isoproterenolhydrochloride, terfenadine, propranolol hydrochloride, desipraminehydrochloride, salmeterol, sildenafil citrate, tadalafil, vardenafil,fenamic acids, Piroxicam, Naproxen, Voltaren (diclofenac), rofecoxib,ibuprofen ondanstetron, sumatriptan, naratryptan, ergotamine tartrateplus caffeine, methylsegide, olanzapine, raloxifene, and fenofibrate.11. The method of any preceding claim, wherein the grinding compound isselected from the group consisting of sodium hydrogen sulfate, sodiumhydrogen carbonate, sodium hydroxide, succinic acid, fumaric acid,maleic acid, tartaric acid, citric acid, ammonium chloride, methylaminehydrochloride, ammonium bromide, crystalline hydroxides, hydrogencarbonates, hydrogen carbonates of pharmaceutical acceptable alkalimetals, sodium sulphate, sodium chloride, sodium metabisulphite, sodiumthiosulphate, ammonium chloride, Glauber's salt, ammonium carbonate,sodium bisulphate, magnesium sulphate, potash alum, potassium chloride,sodium carbonate, sodium bicarbonate, potassium carbonate, potassiumbicarbonate, and lactose.
 12. The method of any preceding claim, furthercomprising the step of removing at least a portion of the at leastpartially milled grinding compound, wherein the nanoparticles have anaverage particle size of less than 200 nm.
 13. The method of claim 12,wherein at least 25% of the at least partially milled grinding compoundis removed.
 14. The method of claim 13, wherein at least 50% of the atleast partially milled grinding compound is removed.
 15. The method ofclaim 14, wherein at least 75 of the at least partially milled grindingcompound is removed.
 16. The method of claim 15, wherein substantiallyall of the at least partially milled grinding compound is removed.
 17. Amethod for manufacturing a medicament comprising the method of any ofclaims 1-16, and further comprising the step of combining atherapeutically effective amount of the nanoparticle compositionproduced thereby with a pharmaceutically acceptable carrier.
 18. Ananoparticle composition comprising nanoparticles of a biologicallyactive compound produced by the method of any of claim 1-16.
 19. Apharmaceutical composition comprising nanoparticles of a biologicallyactive compound produced by the method of claim
 17. 20. The nanoparticlecomposition of claim 18, wherein the nanoparticles have an average sizeless than a size selected from the group consisting of 200 nm, 100 nm,75 nm, 50 nm, and 40 nm.
 21. The nanoparticle composition of claim 20,wherein the particle size of at least 50% of the nanoparticles is withinthe average size range.
 22. The nanoparticle composition of claim 21,wherein the particle size of at least 75% of the nanoparticles is withinthe average size range.
 23. The pharmaceutical composition of claim 19,wherein the nanoparticles have an average size less than a size selectedfrom the group consisting of 200 nm, 100 nm, 75 nm, 50 nm, and 40 nm.24. The pharmaceutical composition of claim 23, wherein the particlesize of at least 50% of the nanoparticles is within the average sizerange.
 25. The pharmaceutical composition of claim 24, wherein theparticle size of at least 75% of the nanoparticles is within the averagesize range.
 26. The nanoparticle composition of any of claims 20-22,wherein the biologically active compound is diclofenac.
 27. Thenanoparticle composition of claim 26, wherein the grinding compound isat least one member selected from the group consisting of Na₂CO₃,NaHCO₃, NH₄Cl, and NaCl.
 28. The pharmaceutical composition of any ofclaims 23-25, wherein the biologically active compound is diclofenac.29. The pharmaceutical composition of claim 28, wherein the grindingcompound is at least one member selected from the group consisting ofNa₂CO₃, NaHCO₃, NH₄Cl, and NaCl.
 30. The nanoparticle composition of anyof claims 20-22, wherein the biologically active compound is naproxen.31. The nanoparticle composition of claim 30, wherein the grindingcompound is at least one member selected from the group consisting ofNa₂CO₃, NaHCO₃, NH₄Cl, and NaCl.
 32. The pharmaceutical composition ofany of claims 23-25, wherein the biologically active compound isnaproxen.
 33. The pharmaceutical composition of claim 32, wherein thegrinding compound is at least one member selected from the groupconsisting of Na₂CO₃, NaHCO₃, NH₄Cl, and NaCl.
 34. The nanoparticlecomposition of any of claims 20-22, wherein the biologically activecompound is olanzapine.
 35. The nanoparticle composition of claim 34,wherein the grinding compound is NH₄Cl.
 36. The pharmaceuticalcomposition of any of claims 23-25, wherein the biologically activecompound is olanzapine.
 37. The pharmaceutical composition of claim 36,wherein the grinding compound is NH₄Cl.
 38. The nanoparticle compositionof any of claims 20-22, wherein the biologically active compound issildenafil.
 39. The nanoparticle composition of claim 38, wherein thegrinding compound is citric acid.
 40. The pharmaceutical composition ofany of claims 23-25, wherein the biologically active compound issildenafil.
 41. The pharmaceutical composition of claim 40, wherein thegrinding compound is citric acid.
 42. The nanoparticle composition ofany of claims 20-22, wherein the biologically active compound israloxifene.
 43. The nanoparticle composition of claim 42, wherein thegrinding compound is NaCl.
 44. The pharmaceutical composition of any ofclaims 23-25, wherein the biologically active compound is sildenafil.45. The pharmaceutical composition of claim 44, wherein the grindingcompound is NaCl.
 46. A nanoparticle composition comprisingnanoparticles of a biologically active compound, formed by the processof dry milling a solid biologically active compound and a millablegrinding compound in a mill comprising a plurality of milling bodies,for a sufficient time period to produce a solid dispersion comprisingnanoparticles of the biologically active compound dispersed in at leastpartially milled grinding compound.
 47. The nanoparticle composition ofclaim 46, wherein the nanoparticles have an average size than a sizeselected from the group consisting of 200 nm, 100 nm, 75 nm, 50 nm, and40 nm.
 48. The nano particle composition of claim 47, wherein theparticle size of at least 50% of the nanoparticles is within the averagesize range.
 49. The nanoparticle composition of claim 48, wherein theparticle size of at least 75% of the nano particles is within theaverage size range.
 50. The nanoparticle composition of any of claims46-49, wherein the process further comprises the step of removing atleast a portion of the at least partially milled grinding compound. 51.A method of treating a human in need of such treatment comprising thestep of administering a pharmaceutically effective amount of a memberselected from the group consisting of the nanoparticle composition ofany preceding claim, the pharmaceutical composition of any precedingclaim, and the medicament of any preceding claim.